GIFT  OF 


CAROTIN 

THE  PRINCIPAL  NATURAL  YELLOW 
PIGMENT  OF  MILK  FAT 


CHEMICAL    AND    PHYSIOLOGICAL    RELATIONS    OF 

PIGMENTS  OF  MILK  FAT  TO  THE  CAROTIN 

AND  XANTHOPHYLLS  OF  GREEN 

PLANTS 


BY 


LEROY  SHELDON  PALMER,  B.S.  in  Ch.E.,  M.A. 

SUBMITTED  IN  PARTIAL  FULFILLMENT  OF  THE 

REQUIREMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 


IN  THE 


GRADUATE  SCHOOL 

OF  THE 

UNIVERSITY  OF  MISSOURI 


1913 


CAROTIN 

THE  PRINCIPAL  NATURAL  YELLOW 
PIGMENT  OF  MILK  FAT 


CHEMICAL    AND    PHYSIOLOGICAL    RELATIONS    OF 

PIGMENTS  OF  MILK  FAT  TO  THE  CAROTIN 

AND  XANTHOPHYLLS  OF  GREEN 

PLANTS 


BY 

LEROY  SHELDON  PALMER,  B.S.  in  Ch.E,  M.A. 

SUBMITTED  IN  PARTIAL,  FULFILLMENT  OF  THE 

REQUIREMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 


IN  THE 


GRADUATE  SCHOOL 

OF  THE 

UNIVERSITY  OR  MISSOURI 


1913 


TABLE  OF  CONTENTS 


PART  I— HISTORICAL 

PAGE 

I.  INTRODUCTION 313 

1.  The  Pigment  of  Butterfat  as  a  Factor  in  the  Colora- 

tion of  Milk 316 

2.  Object  of  Present  Investigations 316 

II.  HISTORICAL. 

1.  The  Pigments  of  Plant  Origin. 

(a)  The  Carotins. 

(1)  The  Pigment  of  the  Carrot , 317 

(2)  The  Carotin  of  Green  Plants 318 

(3)  Carotin  in  Flowers,  Fruits  and  Seeds   . .  321 

(b)  The  Xanthophylls 322 

2.  The  Pigments  of  Animal  Origin. 

(a)  The  Luteins 324 

(b)  The  Chromophanes 325 

(c)  The  Lipochromes   326 

(1)  The    General    Properties    of    the    Lipo- 

chromes    328 

(2)  The  Lipochromes   of   Algae,    Fungi,   and 

Bacteria  328 

(3)  The  Lipochrome  of  Egg  Yolk 329 

3.  The  Physiological  Relation  Between  Plant  and  Ani- 

mal   Lipochromes 330 

III.  SUMMARY    332 

IV.  BIBLIOGRAPHY   334 

PART  II— RELATIONS  OF  MILK  FAT  PIGMENTS  TO  CARO- 
TIN AND  XANTHOPHYLLS  OF  GREEN  PLANTS 

I.     INTRODUCTION 339 

II.     METHODS  OF  ISOLATION 340 

1.     General  Properties  of  Butter  Fat  Pigments 341 

3 


S99518 


PAGE 

III.  METHODS  OF  IDENTIFICATION 342 

1.  Relative  Solubility  of  Carotin  and  Xanthophylls 342 

2.  Adsorption  Properties  of  Carotin  and  Xanthophylls  . .   344 
(a)     Carotin  and  Xanthophylls  of  Green  Plants  ....  346 

IV.  IDENTIFICATION  OF  BUTTER  FAT  PIGMENTS 350 

1.     Standardization  of  Absorption  Bands 355 

V.     CHARACTER  OF  PIGMENTS  IN  DIFFERENT  BUTTER  FATS  . . .  356 

1.     Light  Colored  Fats.     2.    After  Carrot  Feeding 357 

3.  Colostrum  Milk  Fat  358 

4.  Crystalline  Form  of  Carotin  from  Butter  Fat 359 

VI.     RELATION  BETWEEN  COLOR  OF  MILK  FAT  AND  FOOD  OF  Cow  360 

1.  Yellow  Pigments  of  Common  Cattle  Feeds 360 

(a)  Cottonseed  Meal  and  Cottonseed  Hulls 361 

(b)  Bleached  Alfalfa  Hay  362 

(c)  Yellow  Corn 362 

(d)  The  Carrot   362 

2.  The  Feeding  Experiments 364 

VII.     RELATION  BETWEEN  COLOR  OF  MILK  FAT  AND  BREED  OF 

Cow   377 

VIII.     DISCUSSION  OF  RESULTS  383 

IX.     SUMMARY  386 

X.     BIBLIOGRAPHY     387 

PART  III— PIGMENTS  OF  BODY  FAT,  CORPUS  LUTEUM 
AND  SKIN  SECRETIONS  OF  THE  COW 

I.     INTRODUCTION    391 

II.     METHODS  OF  IDENTIFICATION 392 

III.     THE  PIGMENTS  OF  THE  BODY  FAT 394 

1.  Methods  of  Isolation  395 

2.  Identification  of  Pigments 395 

(a)  Experiment  I 395 

(b)  Experiment  II    396 

4 


PAGE 

IV.     RELATION  BETWEEN  COLOR  OF  BODY  FAT  AND  FOOD  OF 

Cow 397 

V.     RELATION  BETWEEN  COLOR  OF  BODY  FAT  AND  BREED  OF 

Cow   399 

VI.  THE  PIGMENTS  OF  THE  CORPUS  LUTEUM  402 

VII.  THE  PIGMENTS  OF  WAXY  SECRETIONS  OF  JERSEY  Cows  . .  405 

VIII.     BODY  FAT  AND  BLOOD  SERUM  PIGMENTS  OF  NEW-BORN 

CALF    406 

IX.     DISCUSSION  OF  RESULTS 408 

X.     SUMMARY 410 

XL     BIBLIOGRAPHY   411 

PART  IV. 
A.  THE  YELLOW  PIGMENT  OF  BLOOD  SERUM 

I.     INTRODUCTION    415 

II.     METHODS  OF  IDENTIFICATION 416 

III.  METHODS  OF  ISOLATION    417 

IV.  CHEMICAL  IDENTIFICATION  OF  PIGMENT 418 

V.     DISCUSSION  OF  EXPERIMENTS 421 

VI.  PHYSIOLOGICAL  RELATION   BETWEEN   CAROTIN  OF   BLOOD 

SERUM  AND  FOOD  OF  Cow 422 

VII.  TRANSPORTATION   OF   CAROTIN    AND   XANTHOPHYLLS    BY 

BLOOD 427 

VIII.     THE  HIGH  COLOR  OF  COLOSTRUM  MILK  FAT 435 

5 


PAGE 

IX.     DISCUSSION  OF  RESULTS  437 

X.     SUMMARY    438 

XI.     BIBLIOGRAPHY     439 

B.    CAROTIN  AND  XANTHOPHYLLS  DURING  DIGESTION 

I.  INTRODUCTION 441 

II.  METHODS  OF  STUDY 441 

III.  ACTION  OF  DIGESTIVE  JUICES 442 

IV.  CHARACTER  OF  PIGMENTS  ALONG  DIGESTIVE  TRACT 444 

V.     THE  EXCRETED  PIGMENTS 445 

VI.  DISCUSSION  OF  RESULTS 445 

VII.  SUMMARY  AND  BIBLIOGRAPHY 446 

C.    THE  PIGMENTS  OF  HUMAN  MILK  FAT 

I.     INTRODUCTION    447 

II.  EXPERIMENT  I 447 

III.  EXPERIMENT  II    448 

IV.  DISCUSSION  OF  RESULTS   449 

V.  SUMMARY  .                                                                               .  450 


BIOGRAPHY  451 


CAROTIN— THE  PRINCIPAL  NATURAL  YELLOW  PIGMENT  OF 

MILK  FAT. 


Its  Relations  to  Plant  Carotin  and  the  Carotin  of  the  Blood  Serum, 
Body  Fat  and  Corpus  Luteum. 


LEROY  S.  PALMER  AND  C.  H.  ECKLES. 

The  investigations  dealing  with  the  natural  yellow  pigment  of 
milk  fat  will  appear  as  a  series  of  four  bulletins  as  follows : 

Part.  I.  A  Review  of  the  Literature  Concerning  the  Yellow  Plant 
and  Animal  Pigments.  Missouri  Agricultural  Experiment 
Station  Research  Bulletin  No.  9. 

Part  II.  The  Chemical  and  Physiological  Relation  of  the  Pigments 
of  Milk  Fat  to  the  Carotin  and  Xanthophylls  of  Green  Plants. 
Missouri  Agricultural  Experiment  Station  Research  Bulletin 
No.  10. 

Part.  III.  The  Pigments  of  the  Body  Fat,  Corpus  Luteum  and 
Skin  Secretions  of  the  Cow.  Missouri  Agricultural  Experi- 
ment Station  Research  Bulletin  No.  n. 

Part  IV.     (A).  The  Yellow  Lipochrome  of  Blood  Serum.     (B.) 
The  Fate  of  Plant  Carotin  and  Xanthophylls  During  Diges- 
tion.    (C).  The  Pigments  of  Human  Milk  Fat.   Missouri  Ag- 
ricultural Experiment  Station  Research  Bulletin  No.  12. 
The  present  paper  is  the  first  of  the  series.    As  indicated  it  will 
"be  confined  entirely  to  a  review  of  the  extensive  literature  in  regard 
to  the  yellow  plant  and  animal  pigments. 

Part  II  will  be  a  report  of  the  chemical  identification  of  the  milk 
fat  pigment.  It  will  also  include  a  number  of  investigations  showing 
the  relation  between  the  amount  of  pigment  in  the  milk  fat  and  the 
character  of  the  ration  and  the  breed  of  the  cow. 

Part  III  will  consist  of  the  data  showing  the  chemical  identification 
of  the  pigments  mentioned.  Data  will  also  be  presented  showing  the 
relation  between  the  color  of  the  body  fat  and  the  character  of  the 

(313) 


314        MISSOURI  :AG$.^EXF.  STA.  RESEARCH  BULLETIN  No.  9. 


ration  and  the  breed  of  the  cow.  A  brief  experiment  will  also  be 
reported  showing  the  absence  of  these  pigments  in  the  body  of  a  new- 
born Jersey  calf. 

Part  IV  (A)  will  report  the  chemical  identification  of  the  blood 
serum  pigment.  It  will  show  how  blood  carries  the  pigment  and  what 
effect  the  character  of  the  ration  has  upon  the  amount  of  pigment 
carried  by  the  blood  and  the  amount  secreted  in  the  milk  at  the  same 
time.  A  brief  study  of  the  cause  of  the  high  color  of  colostrum 
milk  will  also  be  reported.  (B)  This  will  consist  of  the  report  of  a 
few  investigations  relative  to  the  fate  of  the  carotin  and  xanthophyll? 
of  plants  during  their  passage  through  the  cow's  body.  (C)  The 
experiments  reported  here  will  show  the  character  of  the  pigments 
of  human  milk  fat. 


CAROTIN,  THE  PRINCIPAL  YELLOW,  P^tfMEN'rpf  Miiac  FAT.    315 


A  REVIEW  OF  THE  LITERATURE  CONCERNING  THE  YELLOW 
PLANT  AND  ANIMAL  PIGMENTS. 

It  has  been  the  custom  for  generations  to  judge  the  quality  of 
dairy  products  to  a  large  extent  by  their  yellow  color.  This  has  been 
carried  to  such  an  extent  that  the  manufacturer  of  butter,  whether 
it  be  on  a  large  or  small  scale,  finds  it  impossible  to  market  butter  that 
does  not  have  a  standard  yellow  color.  The  consumer  of  milk  or  cream 
as  a  rule  looks  upon  a  yellow  color  as  indicating  the  richness  and  quality 
of  the  product.  Although  it  is  well  known  that  the  color  has  no 
relation  to  the  food  value  of  milk  or  cream,  the  popular  prejudice 
is  so  strong  that  the  producer  of  market  milk  has  to  take  it  into 
account  and  try  to  supply  a  product  with  as  much  natural  yellow 
color  as  possible. 

During  part  of  the  year,  namely  during  the  spring  and  early 
summer  and  usually  also  in  the  early  fall,  the  fresh  green  feeds  which 
the  cows  receive  give  the  shade  of  yellow  to  the  milk  fat  which  the 
consumer  demands.  During  the  winter  months,  or  in  summer  if  the 
pastures  become  dry,  this  yellow  color  is  wholly  or  in  part  absent 
from  the  milk  fat,  and  the  butter  manufacturer  is  then  forced  to 
color  the  butter  artificially,  in  order  to  maintain  the  required  standard. 

It  is  generally  accepted  as  a  fact  that  the  breed  of  the  cow  has 
a  pronounced  relation  to  the  color  of  the  milk  fat  and  that  the  Guernsey 
and  Jersey  breeds  rank  first  in  this  respect.  The  breeders  of  this  class 
of  cattle  have  emphasized  this  characteristic  as  one  of  the  strong  points 
of  their  respective  breeds.  This  characteristic  of  Guernsey  and  Jersey 
breeds,  as  compared  with  the  Holstein  and  Ayrshire  breeds,  has  been 
generally  attributed  to  physiological  differences.  According  to  this 
view,  Guernsey  and  Jersey  cattle  are  able  to  produce  a  higher  colored 
fat  due  to  some  inherent  quality,  just  as  they  are  able  to  produce  a 
higher  percentage  of  fat  in  their  milk.  It  is  a  well-known  fact  that 
the  skin  and  the  secretions  of  the  skin  of  Guernsey  and  Jersey  cattle 
have  a  higher  yellow  color  than  other  breeds,  and  this  characteristic 
is  looked  upon  by  cattle  breeders  as  an  indication  of  the  ability  of 
animals  of  these  breeds  to  produce  highly  colored  milk  fat. 

The  body  fat  of  Guernsey  and  Jersey  cattle  is  also  characterized 
by  a  high  yellow  color  and  for  this  reason  beef  from  these  animals 
is  often  looked  upon  with  disfavor  by  the  butcher  and  the  consumer. 

That  the  yellow  color  of  butter  has  a  relation  to  its  market  value 
is  shown  by  the  fact  that  "color"  has  a  place  on  the  standard  butter 
score  cards  with  a  value  of  fifteen  out  of  one  hundred  points.  The 


316        Mis£oOtfi;A^k.«^i>;  STA:  RESEARCH  BULLETIN  No.  9. 

oleomargarin  manufacturers  have  also  recognized  the  value  of  color 
and,  so  far  as  the  law  has  permitted,  have  made  a  practice  of  coloring 
oleomargarin  in  imitation  of  butter.  When  the  law  placed  a  tax  on 
artificially  colored  oleomargarin,  or  in  some  cases  prohibited  it  entirely, 
the  manufacturers  began  using  only  the  highest  colored  beef  fats 
that  could  be  bought  or  mixed  the  oleomargarin  with  butter  having 
a  high  natural  color,  in  order  to  produce  the  color  they  sought. 

The  Pigment  of  the  Butter  Fat  as  a  Factor  in  the  Coloration  of  Milk. 

The  more  or  less  yellow  color  of  cows'  milk  which  is  especially 
evident  in  the  cream  and  butter  has  not  been  attributed  in  all  cases 
to  the  same  pigment.  On  the  one  hand  a  few  authors  have  stated 
that  the  pigment  of  butter  is  manifested  in  the  familiar  yellow  color 
of  milk  whey.  This  view  originated  with  Blyth  l  who  called  the  whey 
pigment  lactochrome  and  the  view  has  found  its  way  into  a  number 
of  texts.  On  the  other  hand  a  larger  number  of  authors  have  ignored 
the  whey  pigment  and  considered  the  lipochrome-like  pigment  of  the  milk 
fat  to  be  the  only  factor  causing  the  yellow  color  of  cream  and  butter. 

The  investigations  which  were  carried  on  in  this  laboratory  have 
been  the  first  to  point  out  that  the  whey  pigment  and  the  butter  fat  pig- 
ment are  not  identical  but  are  distinct  substances ;  and  that  both  are  of 
importance  in  causing  the  yellow  color  of  milk.  The  pigment  of  the 
butterfat  is  the  more  important  of  the  two,  however.  The  pigment 
of  the  whey  is  of  secondary  importance,  and  is  of  an  entirely  different 
nature.  Its  probable  identity  with  urochrome,  the  specific  urinary  pig- 
ment, has  recently  been  shown  by  one  of  us.2 

Object  of  the   Present   Investigations. 

The  present  investigations  were  undertaken  primarily  to  study  the 
chemical  nature  of  the  yellow  butterfat  pigment  and  to  classify  it  from 
a  scientific  standpoint.  At  the  same  time  information  was  gathered 
with  the  hope  of  ascertaining  to  what  extent  the  generally  accepted 
views  concerning  the  color  of  milk  fat  are  correct  in  order  to  establish 
a  scientific  basis  for  the  subject  which  would  be  of  value  to  those 
interested  in  the  handling  of  dairy  products  in  a  commercial  way. 

In  the  principal  part  of  the  investigation  it  was  sought;  (i)  to 
show  the  chemical  and  if  possible  the  physiological  relation  of  the 
butter  fat  pigment  to  similar  animal  pigments  such  as  the 

1.  A.   W.   Blyth,   "Foods.     Their   Composition   and   Analysis"   Text,   4th 
Edition  1896,  p.  239. 

2.  Lactochrome:     The  Yellow  Pigment  of  Milk  Whey,  etc.,  by  Leroy  S. 
Palmer  and  Leslie  H.  Cooledge.     Missouri  Agricultural  Experiment  Station 
Research  Bulletin  No.  13;;  Jour.  Biol.  Chem.  XVII,  p.  251  (1914). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    317 

corpus  luteum  pigment,  the  body  fat  pigment,  and  the  blood  serum 
pigment;  and  (2)  to  show  the  chemical  and  physiological  relation  of 
the  butterfat  pigment  to  the  carotin  and  xanthophylls  of  green  plants. 
In  the  secondary  part  of  the  investigation  it  was  sought  to  study 
the  influence  of  certain  factors  which  have  both  practical  and  scientific 
bearing  upon  the  color  of  the  butterfat,  among  which  are  the  breed 
of  the  animal  and  the  character  of  the  ration,  the  latter  in  connection 
with  the  chemical  and  physiological  studies  indicated  above. 

THE   PIGMENTS  OF   PLANT  ORIGIN. 

The  earliest  researches  on  plant  pigments  dealt  with  the  green 
pigments.  Caventon  first  called  them  chlorophyll  in  1817.  His  work, 
however,  was  preceded  by  the  pioneers  in  this  field,  among  which  the 
names  of  Grew,  whose  work  is  dated  1682,  and  Rouelle,  Meyer, 
Fourcroy,  Berthellot,  Senebier,  Proust  and  Vanquelin  are  of  historical 
interest. 

The  Carotins. 

The  Pigment  of  the  Carrot.  The  yellow  pigment  of  the  cultivated 
carrot  (Daucus  Carota)  has  long  been  of  interest  to  botanists  and 
chemists,  the  investigations  of  this  body  having  extended  over  almost 
one  hundred  years. 

Wachenroder  1  was  the  first  investigator  of  the  carrot  pigment. 
He  isolated  it  and  called  it  Karotin.  The  work  of  Vanquelin  and 
Bouchardat  2  soon  followed  and  a  little  later  Zeise  3  took  up  the  study. 
He  obtained  the  first  crystals  and  assigned  to  them  the  chemical  formula 
C5  H10  or  10  (C5  H8). 

Husemann 4  was  the  next  investigator.  He  found  six  per  cent 
of  oxygen  in  his  pure  preparation  and  gave  the  pigment  the  formula 
C18  H24  O.  A  secondary  pigment  which  he  thought  always  accom- 
panied the  carotin  in  small  amounts,  he  named  hydrocarotin  and  gave 
it  the  formula  C18  H30  O. 

It  is  to  Arnaud 5  however  that  we  are  indebted  for  the  first 
thorough  research  in  regard  to  the  carrot  pigment  carotin.  The  crys- 
tals which  he  obtained  were  flat,  rhombic-shaped  crystals,  red  orange 
by  transmitted  light,  and  greenish  blue  by  reflected  light.  They  melted 
at  168°  C.  He  showed  beyond  a  doubt  that  the  pigment  was  simply 

1.  Dissertatio  de  Anthelminticis  Gottingen  1826 ;  also  Geigers  Magaz.  Pharm. 
33  p.  144  (1831);  also  Berzelius  Jahresber.  12  p.  277  (1833). 

2.  Schweizg.  Jour.  Chem.  58,  p.  95  (1830). 

3.  Lieb.  Ann.  62  p.  380  (1847);  Annal.  Chem.  Phys.  (3)  20,  p.  125  (1847). 

4.  Lieb.  Annal.  117  p.  200  (1860). 

5.  Compt  Rend.  102  p.  1119   (1886),  p.  1319  (1887);  Jour.  Pharm.  Chim. 
14  p.  149    (1886). 


318        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

an  unsaturated  hydrocarbon.  He  gave  it  the  formula  C26  H38  and  the 
iodine  derivative  the  formula  C26  H38  I2. 

Eiiler  and  Nordenson  1  report  the  most  recent  investigations  in 
regard  to  the  carrot  pigment.  They  found  their  crystalline  prepara- 
tion to  be  mixed  with  crystals  of  xanthophyll ;  they  also  showed  that 
the  belief  often  advanced  that  carotin  is  chemically  related  to  choles- 
terol, is  unfounded. 

The  Carotin  of  Green  Plants. — Arnaud  2  was  one  of  the  first  in- 
vestigators to  show  that  the  carrot  carotin  is  identical  in  properties 
with  a  yellow  constituent  of  chlorophyll,  although  the  existence  of 
this  yellow  constituent  of  chlorophyll  had  long  been  the  subject  of 
investigation. 

Berzelius  3  first  sought  to  isolate  a  yellow  pigment  from  autumn 
leaves  by  extracting  with  alcohol.  He  called  it  "Blattgelb"  or  xantho- 
phyll, and  expressed  the  belief  that  the  pigment  pre-existed  along  with 
the  green  coloring  matter  of  the  leaf. 

The  subject  subsequently  received  the  attention  of  many  investi- 
gators. Fremy,4  Michels,  Millardet,  Miiller,  Tinisnsseff,  Gerland,  Ran- 
nenhoff,  Askennasy,  Stokes,  Sorby,5  Tschirch,6  Kraus,7  Filhol,8 
Hansen,9  Conrad,10  Wiesner,11  and  many  others  took  up  the  investiga- 
tion. 

Fremy  designated  the  yellow  pigment  Phylloxanthin.  Filhol 
noticed  that  by  treating  crude  alcoholic  chlorophyll  solutions  with  ani- 
mal charcoal  it  was  possible  to  remove  the  green  constituent  of  the 
mixture  leaving  a  yellow  colored  solution,  the  color  of  which  he  be- 
lieved was  due  to  a  pre-existing  pigment  or  pigments  associated  with 
the  green  one.  Kraus  confirmed  the  observations  of  Filhol,  and  was 
the  first  to  notice  that  when  an  alcoholic  solution  of  chlorophyll  is 
shaken  with  benzoline  (petroleum  ether)  the  alcohol  retains  the  yellow 
coloring  matter,  the  benzoline  taking  up  the  green  constituent.  Kraus' 
investigation  was  also  the  first  to  show  that  the  ordinary  chlorophyll 
spectrum  was  due  partly  to  the  green  and  partly  to  the  yellow  con- 
stituent, which  he  called  xanthophyll.  Kraus'  xanthophyll  gave  a 

1.  Zeit.  f.  Physiol.  Chem.  56,  p.  223  (1908). 

2.  Compt  Rend.  100,  p.  751  (1885);  104  p.  1293  (1887). 

3.  Ann.  d.  Chem.  21,  p.  257  (1837). 

4.  Ann.  Sc.  Nat.  13,  p.  45  (1860);  Compt.  Rend.  41,  p.  189  (1865). 
6.  Proc.  Roy.  Soc.  21,  p.  456   (1875). 

6.  Botan.  Zeitung.  42,  p.  817  (1884). 

7.  Flora,  p.  155  (1875). 

8.  Compt.  Rend.  39,  pp.  9-184;  50,  pp.  545  and  1182. 

9.  Sitz.  ber.  d.  phys.  Med.  Ges.  Wiirzberg  (1883);  and  Arbeiten  d.  Botan. 
Gessel.  Wurzberg,  3,  p.  127  (1884)  and  "Die  Farbstoff  des  Chlorphylls"  (1889). 

10.  Flora,  Vol.  25  (1872). 

11.  Flora,  Vol.  (1874);  Sitz.  der.  Wein.  Akad.  89,  1.  abts.  p.  325. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    319 

dark  blue  coloration  with  concentrated  H2  SO4,  and  bleached  very 
quickly  in  the  sunlight. 

Sorby,  using  carbon  bisulphide  as  the  separator  in  place  of  benzo- 
line,  was  the  first  to  show  that  there  is  more  than  one  yellow  pigment 
associated  with  chlorophyll. 

Hansen's  method  of  isolating  the  yellow  pigments  was  still  dif- 
ferent. He  treated  the  alcoholic  extracts  with  caustic  alkali,  evaporated 
the  liquor  to  dryness  and  extracted  the  yellow  pigment  from  the 
residue  with  ether,  the  spectroscopic  study  of  which  led  him  to  believe 
that  it  exhibited  three  absorption  bands.  He  believed  also  that  it  was 
identical  with  the  pigment  of  the  carrot. 

E.  Schunck 1  obtained  from  all  crude  alcoholic  chlorophyll  ex- 
tracts minute  sparkling  red  crystals  which  deposited  on  standing,  and 
which  he  considered  identical  with  the  crystals  which  Bougarel  2  had 
called  erythrophyll,  and  which  Hartsen3  has  called  crysophyll.  This 
pigment  showed  two  absorption  bands. 

Tschirch 4  using  Hansen's  method,  found  two  yellow  coloring 
matters,  to  which  he  gave  the  name  xantho-carotin,  showing  three 
bands,  and  xanthophyll  proper  which  showed  no  bands. 

Returning  now  to  Arnaud's  5  work,  we  find  that  he  identified 
the  red  orange  crystalline  pigment  which  he  obtained  from  spinach 
leaves  with  the  carotin  of  the  carrot,  both  as  regards  to  crystalline 
form,  melting  point  and  chlorine  derivatives. 

We  are  indebted  to  Immendorff  6  for  the  confirmation  of  Arnaud's 
results  indicating  that  the  carotin  of  green  plants  is  identical  with 
the  carotin  of  the  carrot.  Immendorff  gave  the  pigment  the  formula 
which  Arnaud  found  for  carotin,  namely  C26  H38.  He  states,  however, 
that  the  percentage  composition  of  the  pure  pigment  corresponded  best 
with  Zeise's  formula,  C5  H8.  Immendorff  believed  that  carotin  was 
the  only  yellow  pigment  accompanying  chlorophyll  in  the  green  leaf. 

One  of  the  most  extensive  publications  in  regard  to  carotin  is 
that  by  F.  G.  Kohl 7  This  author  also  gives  one  of  the  best  and  most 
voluminous  compilations  of  the  carotin  literature  that  is  to  be  found, 
besides  a  large  amount  of  experimental  data.  The  literature  is  also 
excellently  reviewed  by  Tammes.8  Kohl  gave  carotin  the  formula 

1.  Proc.  Roy.  Soc.  44,  p.  449. 

2.  Ber.  Chem.  Gessel,  10,  p.  1173  (1877). 

3.  Arch.  Pharm.  207,  p.  166  (1875). 

4.  Botan.  Zeitung.  42,  p.  817  (1884);  Ber.  der  Deutsch.  Botan.  Ges.  14,  pt. 
2,  p.  76  (1896). 

5.  Compt  Rend.  100,  p.  751  (1885). 

6.  Landwirtschaftliche  Jahrbiicher  18,  p.  507  (1889). 

7.  Untersuch,  Tiber  d.  Karotin,  Leipzig.  1902. 

8.  Flora,  p.  205  (1900). 


320        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

C26  H38,  and  the  iodine  derivative  C26  H38 I2.  He  also  gave  a  detailed 
description  of  the  spectroscopic  absorption  of  carotin.  In  ether  and 
carbon-bisulphide  he  measured  three  bands : 

In  ether  In  carbon  bisulphide 

I     49<>475  A  I     510-485  * 

II    455-445  *  II    470-458  * 

III    430-418  ^  III    437-425   A 

Carotin  is  laevorotatory,  according  to  Kohl,  «D  at  15°  in  chloroform 
being— 30.17°. 

Schunck  *  in  his  spectroscopic  study  of  the  yellow  pigments  of 
leaves  and  flowers,  described  the  properties  of  carotin.  Schunck  also 
photographed  the  absorption  bands  of  crysophyll  (carotin)  from  the 
daffodil  leaf,  from  spinach,  from  the  carrot  and  from  grass,  in  alcoholic 
solution.  All  of  the  carotin  preparations  showed  the  same  three  pro- 
nounced bands  situated  between  F  and  H  the  first  band  of  which 
lay  almost  directly  upon  the  F  line. 

The  most  recent  detailed  investigation  of  the  carotin  of  green 
plants  is  that  of  Willstatter  2  and  Meig,  and  a  study  of  their  data  shows 
that  their  results  are  to  be  accepted  as  the  final  proof  of  the  chemical 
constitution  and  properties  of  this  pigment. 

Willstatter  and  Meig  describe  the  properties  of  carotin  as  follows : 
Its  crystals  are  copper  colored  plates  of  almost  quadratic  form,  and 
melt  at  167.5°  to  168°  C.  Its  crystals  are  soluble  with  great  difficulty 
in  hot  ethyl  alcohol  and  almost  insoluble  in  cold  ethyl  alcohol,  and 
in  methyl  alcohol  they  are  still  less  soluble;  one  gram  of  the  crystals 
requires  1.5  liters  of  petroleum  ether  (b.  p.  30-50  °C.)  for  solution  and 
about  900  c.cm.  of  hot  ethyl  ether ;  the  crystals  are  difficultly  soluble  in 
acetone,  easily  soluble  in  benzol,  very  easily  soluble  in  chloroform 
and  instantly  soluble  in  carbon  bisulphide;  the  crystals  are  soluble  in 
concentrated  sulphuric  acid  with  an  indigo  blue  color  and  are  pre- 
cipitated as  green  flakes  on  dilution  with  water. 

The  carotin  obtained  by  Willstatter  and  Meig  crystallized  from 
its  deep  red  carbon  bisulphide  solution  on  addition  of  absolute  alcohol, 
but  analysis  showed  that  the  crystals  contained  ^2  to  ^  of  a  molecule 
of  alcohol  of  crystallization.  The  carotin  showed  the  composition  of 
a  pure  hydrocarbon  only  after  crystallization  from  low  boiling  point 
petroleum  ether.  From  this  solvent  the  preparation  of  Willstatter 
and  Meig  showed  the  composition  C5  H7.  A  preparation  of  carotin 
which  the  same  authors  obtained  from  the  carrot  showed  the  same 

1.  Proc.  Roy.  Soc.  72,  p.  170  (1903). 

2.  Ann.  der  Chemie,  355,  p.  1  (1907). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    321 

composition.  A  molecular  weight  determination  of  both  the  carotin 
from  the  carrot  and  from  the  Brennessel  leaves,  by  the  ebullioscopic 
method  in  carbon  bisulphide  gave  an  average  of  533  which  corresponds 
exactly  with  8(C5  H7)  or  C40  H56.  This  shows  that  Arnaud's  formula 
of  C26  H38  is  not  quite  correct^  The  same  difference  is  brought  out 
by  the  analysis  of  the  iodine  derivative  which  Willstatter  and  Meig 
also  prepared. 

The  absorption  bands  of  carotin  were  measured  by  Willstatter  and 
Meig  and  they  coincided  almost  exactly  with  those  given,  for  it  by 
Tschirch  *  and  Monteverde2.  They  did  not  attempt  to  measure  the 
third  band  in  the  violet  which  they  considered  to  be  end  absorption, 
but  measured  only  the  two  bands  in  the  blue  and  indigo  blue. 

Willstatter  and  Meig  Tschirch  Monteverde 

(alcohol  sol.)  (alcohol  sol.)  (petroleum  ether  sol.) 


I     488-470  A  I     487-470  A  I     491-472  A 

II     456-438  A  II     457-439  A  II     461-444  A 

Carotin  in  Flowers,  Fruits  and  Seeds.  According  to  Czapek  3  caro- 
tins have  been  identified  in  many  flowers  by  Hansen,4  Immendorff,5 
Kohl,6  Tammes,7  Hilger,8  and  his  pupils,  Wirth,9  Pabst,10  Kirchner,11 
Ehrung  12  and  Schuler.13 

Among  the  fruits,  Arnaud,14  Passerini,15  Kohl,16  Schunck  17  and 
Montanari 18  have  investigated  the  tomato  pigment  and  believed  it  to 
be  a  carotin.  Its  identity  as  a  truly  isomeric  carotin  has  recently  been 
proved  by  Willstatter  and  Escher.19  Schrotter20  has  shown  that  the 
pigment  of  the  pumpkin  is  in  all  probability  a  carotin  and  Desmoliere  21 
has  identified  carotin  in  the  apricot. 

1.  Ber.  d.  deut.  botan.  Ges.  14,  76  (1896);  22,  414  (1904). 

2.  Acta   Horti.  Petropolitani  XIII  Nr.  9,  123  and  150  (1893). 

3.  Bichemie  der  Pflanzen,  vol.  I,  p.  172,  etc. 

4.  5,  6,  7.     Loc.  cit. 

8.  Botan.  Centr.  57,  p.  335  (1894). 

9.  Dissert.  Erlangen  (1891). 

10.  Arch.  Pharm.  230,  p.  108   (1892). 

11.  Dissert.  Erlangen  1892. 

12.  Botan.  Cent.  69,  p.  154   (1897). 

13.  Dissert.  Erlangen.  1899. 

14.  Compt.  Rend.  102,  p.  1119    (1886). 

15.  Compt.  Rend.  100,  p.  875  (1885). 

16.  Loc.  Cit. 

17.  Proc.  Roy.  Soc.  72,  p.  172  (1903). 

18.  Le  Staz.  sp.  agra.  ital.  37,  p.  909    (1904). 

19.  Zeit.  Physiol.  Chem.  64,  p  74    (1910). 

20.  Vehr.  Zool.  bot.  Gessel.  44,  298  (1895). 

21.  Chem.  Centr.  2,  p.  1001,  1902. 


322        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

Among  the  seeds,  Schunck  1  has  found  the  annatto  pigment  to  be  a 
carotin. 

The  Xanthophylls. 

It  was  mentioned  above  that  it  has  been  found  that  a  second  class 
of  pigments  usually  accompanies  carotin.  Investigations  of  this  class 
of  pigments,  now  called  xanthophylls,  has  not  been  as  extended  as 
that  of  carotin  but  the  constitution  and  properties  of  the  xanthophylls 
are  nevertheless  at  present  established. 

Sorby2  differentiated  the  pigments  accompanying  chlorophyll  as 
xanthophyll,  orange  xanthophyll,  and  yellow  xanthophyll,  all  with 
spectroscopic  properties.  J.  Borodin 3  observed  that  besides  carotin, 
a  second  crystallizable  yellow  substance  exists  in  leaves  which  is  much 
more  soluble  in  alcohol  than  carotin  and  insoluble  in  benzine.  Im- 
mendorff4  denied  the  existence  of  more  than  one  pigment  as  was 
noted  above.  Monteverde5  confirmed  Borodin's  observations. 
Tschirch  6  in  1896,  showed  that  green  leaves  contain  a  second  yellow 
pigment  which,  however,  showed  no  absorption  bands.  Tschirch  called 
the  second  pigment  xanthophyll.  The  name,  however,  was  a  misnomer, 
for  Schunck  7  later  showed  that  Tschirch  was  dealing  with  a  group  of 
water  and  alcohol  soluble  pigments  probably  identical  with  the  lich- 
noxanthine  described  by  Sorby.8  Tschirch9  later  recognized  the  ex- 
istence of  a  true  second  yellow  crystallizable  pigment. 

Molisch  10  in  his  critical  study  of  the  yellow  pigments  left  the 
question  of  their  plurality  an  open  one,  and  Tammes  n  also  left  the 
question  undecided. 

Schunck  12  in  his  widely  known  spectroscopic  study  of  the  yellow 
pigments  of  plants  and  flowers,  demonstrated  beyond  a  doubt  that  a 
second  great  group  of  pigments,  which  he  designates  the  xanthophylls, 
accompanies  the  crysophyll.  He  differentiated  three  different  xantho- 
phylls and  designated  them  L.  B.  and  Y.  xanthophyll,  respectively. 

He  found  that  the  xanthophylls  were  all  characterized  by  giving 
the  same  color  reactions  in  the  dry  state  as  crysophyll  and  three 

1.  Proc.  Roy.  Soc.  72,  1903. 

2.  Proc.  Roy.  Soc.  21,  p.  457  (1875). 

3.  Melanges  Biol.  tir.  d.  bull  d.  L'Acad.  Imp.  d.  St.  Petersb.  11,  p.  512  ( 1883 ) . 

4.  Loc.  cit. 

5.  Loc.  cit.  p.  148   (1903). 

6.  Ber.  d.  d.  Botan.  Gessel,  14,  p.  76  (1896). 

7.  Proc.  Roy.  Soc.  72  (1903). 

8.  Loc.  cit. 

».     Ber.  d.  d.  Botan.  Gessel.  22,  p.  414   (1904). 

10.  "Die  Krystallization  und  der  Nachweis  des  Xanthophylls    (carotins) 
in  Blatte"  (Ber.  d.  Deut.  Botan.  Ges.  14,  p.  18  (1896). 

11.  Loc.  cit.   (1900). 

12.  Proc.  Roy.  Soc.  65  (1899);  68  (1901);  72  (1903.) 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    323 

similar  absorption  bands  in  the  violet  region  of  the  spectrum.  The 
bands  of  the  xanthophylls,  however,  were  all  shifted  somewhat 
towards  the  blue  with  respect  to  the  bands  of  crysophyll,  the  amount 
of  shifting  depending  on  the  xanthophyll,  L  xanthophyll  being  shifted 
the  least  and  Y  xanthophyll  the  most.  Schunck  found  the  absorption 
bands  of  the  different  xanthophylls  especially  characterized  by  the 
action  of  their  alcoholic  solutions  in  the  presence  of  HC1  and  HNO3 
the  details  of  which  are  given  in  his  latest  paper.1 

Schunck  also  made  the  very  interesting  discovery  that  the  yellow 
pigment  of  egg  yolk  and  fowl  serum  shows  the  identical  properties 
of  L  xanthophyll  both  with  respect  to  the  position  of  the  original 
absorption  spectra  and  also  the  action  of  acids  upon  the  spectra.  . 

One  of  the  most  interesting  and  important  studies  of  chlorophyll 
and  its  accompanying  yellow  pigments  was  made  by  Tswett2  who 
discovered  and  thoroughly  investigated  the  adsorption  properties  of 
these  pigments.  He  was  able  to  demonstrate  the  presence  of  at  least 
four  different  xanthophylls  which  he  designates  as  xanthophylls 
*  «'  a"  and  B.  A  more  detailed  review  of  this  work  will  be  given 
in  connection  with  a  report  of  the  present  investigations.  It  is  of 
interest  here  especially  on  account  of  its  historical  position  with  respect 
to  the  establishment  of  the  chemical  constitution  of  the  xanthophylls. 
It  was  Willstatter  and  Meig3  who  isolated  and  identified  the 
crystalline  xanthophyll  pigment  accompanying  the  carotin  in  green 
plants  and  leaves,  and,  as  noted  above,  Eiiler  and  Nordenson4  have 
recently  found  xanthophyll  crystals  in  their  extracts  from  the  carrot, 
thus  indicating  a  more  general  distribution  of  the  xanthophylls  in 
connection  with  carotin  than  has  been  believed. 

The  results  of  the  study  of  the  crystalline  xanthophyll  show  that 
it  is  composed  of  carbon,  hydrogen  and  oxygen  in  the  proportion 
C4t  H56  O2  and  is  thus  merely  carotin  dioxide.5  The  pigment  is  further 
distinguished  from  carotin  by  the  color  and  shape  of  its  crystals,  which 
are  yellow  or  orange  trapesium  plates  sometimes  spear  or  wedge-shaped 
which  are  characterized  by  a  steel  blue  reflection.  The  pigment  ex- 
hibits an  entirely  different  solubility  toward  petroleum  ether  and  abso- 
lute alcohol  than  carotin,  being  insoluble  in  the  former  and  readily 
soluble  in  the  latter  solvent.  According  to  these  authors,  the  pure 

1.  Proc.  Roy.  Soc.  72   (1903). 

2.  Ber.  Botan.  Gessel,  24,  pp.  316  and  384  (1906). 

3.  Ann.  der.  Chemie,  355,  p.  1  (1907). 

4.  Loc.  cit 

5.  Willstatter  and  Meig  point  out  the  probable  identity  of  xanthophyll  with 
the  hitherto  unexplained  hydrocarotin  found  by  Husemann. 


324         MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

crystals  have  a  melting  point  of  172°  C.  (corrected)  which  is  slightly 
higher  than  the  melting  point  of  the  carotin  crystals ;  and  the  absorption 
bands  of  the  pigment  are  slightly  shifted  toward  the  violet  from  the 
corresponding  bands  of  carotin,  as  was  also  shown  by  Schunck  x  for 
the  xanthophylls  which  he  differentiated. 

It  might  be  readily  assumed  that  xanthophyll  is  formed  directly 
from  carotin  in  the  plant.  In  fact  Tschirch  2  has  claimed  that  carrot 
carotin  goes  over  to  xanthophyll  in  the  air.  Eiiler  and  Nordenson  3 
do  not  credit  this  statement  and  state  that,  "One  may  well  suppose 
that  in  the  plant,  xanthophyll  normally  is  formed  from  the  carotin, 
but  outside  of  the  plant  it  has  not  been  possible  to  imitate  this  trans- 
formation, the  most  skillful  oxidation  always  leading  to  a  much 
higher  oxidized  product."  Willstatter  and  Meig  believed  in  this  con- 
nection that  xanthophyll  although  carotin  dioxide  is  not  the  end  pro- 
duct of  the  oxygen  absorption  of  carotin  in  the  plant.  Monteverde  4 
and  Lyubimenko  have  recently  claimed  that  chlorophyll  and  xantho- 
phyll originate  from  the  same  colorless  substance,  carotin  being  a 
complimentary  product  generated  during  the  formation  of  cholorophyll, 
but  not  necessarily  from  the  xanthophyll. 

The  Pigments  of  Animal  Origin. 

The  Luteins. — We  will  now  direct  our  attention  to  a  review  of  the 
literature  bearing  upon  the  yellow  pigments  of  so-called  animal  origin. 
Thudichum  5  was  one  of  the  first  to  investigate  the  yellow  animal  pig- 
ments and  he  classified  a  great  many  of  them  together  with  the  yellow 
pigments  of  plants  under  the  name  lutein,  the  name  being  taken  from 
the  pigment  of  the  corpus  luteum.  He  states,  "Various  parts  of 
animals  and  plants  contain  a  yellow  crystallizable  substance  which  has 
hitherto  not  been  defined,  and  which  I  call  lutein.  It  occurs  in  the 
corpora  lutea  of  the  ovaries  of  animals,  the  serum  of  the  blood,  the 
cells  of  adipose  tissue,  in  butter,  in  the  yolks  of  eggs  of  oviparous 
animals,  in  seeds  such  as  maize,  in  husks  and  pulps  of  fruits  such 
as  annatto,  in  roots  such  as  carrots,  in  leaves  such  as  those  of  coleus, 
and  in  the  stamens  and  petals  of  a  great  many  flowers.'' 

It  is  unfortunate  that  none  of  the  above  statements  are  supported 
by  experimental  evidence,  for  it  can  hardly  be  accepted  that  Thudichum 
was  able  to  obtain  crystals  of  lutein  from  all  the  bodies  in  which  he 

1.  Loc.  cit. 

2.  Ber.  Botan.  Gessel.  22,  p.  414  (1904). 

3.  Loc.  cit. 

4.  Bull.  Acad.  Imper.  Sc.  St.  Petersb.  30,  p.  609   (1912). 

5.  Proc.  Roy.  Soc.  17,  p.  253    (1869). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    325 

claims  to  have  found  it,  or  was  able  to  show  all  the  properties  which 
he  describes  for  the  crystals  which  he  evidently  did  obtain. 

Crystalline  animal  pigments  were  apparently  obtained  before 
Thudichum's  claims  in  this  regard.  According  to  Krukenberg,1  Wittich  2 
obtained  crystals  of  a  red  pigment  from  Euglenia  Sanguirubo,  and 
Piccolo  and  Lieben 3  found  a  crystalline  animal  pigment.  Pouchet  4 
a  little  later  obtained  a  yellow  crystalline  pigment  from  lobsters. 

The  Chromophanes. — The  early  workers  in  the  field  of  animal  pig- 
ments laid  great  emphasis  upon  the  so-called  color  reactions,  one  of 
which,  the  blue  reaction  which  concentrated  HNCX,  was  mentioned  by 
Thudichum.  That  a  similar  reaction  is  given  by  concentrated  H2  SO4 
was  first  noticed  by  Wittich  in  1863,  an<^  Buchholz  also  noticed  it  with 
a  fat  pigment  from  a  Ganglion  cell  of  an  invertebrate.  Piccolo  and 
Lieben  had  also  noticed  the  blue  reaction  with  concentrated  H2  SO4. 
Besides  Thudichum,  Filhol  5  and  Stadeler Q  noticed  the  blue  reaction 
with  concentrated  HNO3.  Stadeler  attempted  to  isolate  the  egg  yolk 
pigment.  He  failed  to  do  so,  however,  but  attempted  to  establish  the 
difference  between  it  and  Bilirubin  with  which  it  bad  been  considered 
identical.  A  little  later  a  third  reaction  of  the  luteins  was  discovered 
by  Schwalbe,7  namely  a  blue-green  color  with  a  solution  of  iodine  in 
potassium  iodide.  Schwalbe  first  noticed  the  reaction  with  the  cone- 
globules  of  the  retinas  of  birds  and  lizard's  eyes.  The  red  globules 
gave  a  beautiful  blue  to  blue-black  color,  and  the  yellow  oil  globules  a 
green  to  blue-green  to  blue.  The  pigments  thus  characterized  were 
called  chromophanes  by  Schwalbe  and  the  existence  of  these  pigments 
was  a  little  later  considerably  extended  by  Capranica  8  who  also  made 
use  of  the  iodine  reaction. 

Kiihne9  took  up  the  study  of  the  chromophanes  of  the  cone- 
globules  of  bird  retinas,  and  separated  three  pigments  which  he  desig- 
nated Rhodophan,  Chlorophan  and  Xanthophan,  respectively,  according 
to  the  color  of  their  solutions. 

Kiihne  also  studied  the  absorption  spectra  and  color  reactions  of 
the  pigment  of  the  egg  yolk  and  the  corpus  luteum  and  compared  them 

1.  Grundzuge   einer   vergleichenden    Physiologie    der   Farbstoff   und    der 
Farben;   1884. 

2.  Arch  f.  Path.  Anat.  27,  p.  573   (1863). 

3.  Giornals  d.  Scienze  Natural!  et.  Economich.  Palermo  2,  p.  258   (1866). 

4.  Jour.  d.  L'Anat  et.  Physiol.  12,  p.  12  (1876). 

5.  Compt.  Rend.  T.  39,  p.  184,  T.  50,  pp.  545  and  1182. 

6.  Jour.  f.  Pract.  Chem.  100,  p.  149  (1867). 

7.  Hand  D.  Ges.  Augenheilkunde  von  Graefe  u.  Saemisch  I,  p.  414  (1874). 

8.  Arch.  f.  Anat.  Physiol.  p.  283   (1877). 

9.  Untersuch,   des   Physiol.   Universitat   Heidelberg   I,    4th   Heft,   p.    341 
(1878);  IV,  p.  169  (1882);  Jour.  Physiol.  1,  p.  109  (1878). 


326        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

with  these  properties  of  the  retinal  pigments.  A  study  of  their  spectro- 
scopic  absorption  properties  led  him  to  believe  that  the  pigments  were 
not  identical. 

Kiihne  in  his  celebrated  work  on  "Optochemie"  occupied  himself 
somewhat  again  with  the  egg  yolk  pigment  and  called  it  Ontochrin 
or  Lecithochrin.  He  did  not  try  to  isolate  it  free  from  nitrogen,  but 
he  did  succeed  in  observing  crystals.  He  again  was  careful  to  dis- 
tinguish between  the  egg  yolk  pigment  and  the  corpus  luteum  pigment, 
which  he  at  this  time  considered  as  extraordinarily  closely  related  to 
carotin. 

The  Lipochromes.  Basing  his  work  on  the  researches  of  Kiihne, 
Krukenberg  commenced  a  series  of  researches  which  extended  from 
1879  to  1886,  the  most  important  of  which  appeared  in  his  "Verglei- 
chende  Physiologische  Studien"  *  and  especially  in  the  paper,  "Grund- 
zuge  einer  vergleichenden  Physiologic  der  Farbstoff  und  der  Farben'r 
which  appeared  in  1884.  Krukenberg  made  an  exhaustive  study  of 
what  had  been  done  on  animal  pigmentation  and  included  under  one 
head  all  those  pigments  which  had  previously  been  known  as  luteins, 
carotin,  zoonerythrin  (tetronerythrin)  and  Kiihne's  chromophanes,  and 
called  them  lipochromes. 

Krukenberg  believed  that  carotin,  the  pigment  of  the  carrot,  was 
the  best  representative  of  the  lipochrome  coloring  matters,  and  ac- 
cepted Husemann's  formula  for  carotin  (C18H24O)  as  representing 
the  chemical  composition  of  the  lipochromes. 

In  regard  to  the  origin  of  lipochromes  Krukenberg  believed,  "It 
is  probable  that  in  most  cases  they  originate  from  fatty  substances, 
for  frequently,  if  not  without  exception,  they  occur  in  company  with 
fat  and  allow  themselves  to  easily  go  over  into  cholesterin-like  bodies." 

In  1885  Krukenberg  2  isolated  a  yellow  lipochrome  from  the  blood 
serum  of  the  ox  by  extracting  the  serum  with  amyl  alcohol.  The 
solution  showed  two  absorption  bands,  one  enclosing  the  line  F  and 
the  other  lying  between  F  and  G.  A  year  later  Halliburton  3  reported 
that  he  extracted  a  yellow  lipochrome  from  the  blood  serum  of  the 
pigeon,  hen,  dove  and  tortoise  by  means  of  alcohol.  Halliburton  re- 
ported an  identical  pigment  in  the  body  fat  of  these  same  animals. 

MacMunn 4  was  the  next  investigator  of  animal  pigments,  and 
like  Krukenberg,  he  extended  the  classification  lipochrome  to  include 

1.  Zoonerythrin  (Tetronerythin) : — Central,  f.  d.  Medic.  Wiss.  1879.    Vergl. 
Physiol.  Studien  I  Reihe,  II  Abth.  s.  67-71;  III  Abth.  s.  114-115;  IV  Abth.  s. 
30-35;  V  Abth.  s.  87-94;  II  Reihe,  I  Abth.  s.  165-167;  III  Abth.  s.  135). 

2.  Sitz,  ber.  d.  Jen.  Gessel.  f.  Med.  1885. 

3.  Jour.  Physiol.  7,  p.  324  (1886). 

4.  Philos  Trans.  Roy.  Soc.  177,  p.  247   (1886). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    327 

the  yellow  constituent  of  chlorophyll  or  Hansen's  "Chlorophyll  Yellow." 
He  believed  that  the  lipochromes  were  chemically  closely  related  to 
chlorophyll.1 

MacMunn's  greatest  contribution  to  animal  chromotology  was  in 
iSSg.2  The  pigments  of  a  great  many  marine  animals,  Crustacea, 
worms  and  sponges  were  examined  and  classified.  Lipochromes  were 
found  abundantly,  MacMunn  drawing  a  distinction  as  to  whether  the 
lipochrome  was  a  rhodophan  or  a  chlorophan-like  lipochrome. 

In  regard  to  some  of  the  properties  of  the  lipochromes  MacMunn 
states,  as  did  Krukenberg,  that  they  are  sensitive  to  light,  both  in  the 
solid  state  and  in  solution,  and  yield  in  many  cases  cholesterin-like 
substances.  He  believed  that  many  of  the  plant  lipochromes  were 
identical  with  the  animal  lipochromes. 

It  will  be  remembered  that  for  a  long  time  there  were  many 
followers  of  the  view  that  a  close  relationship  existed  between  carotin 
and  cholesterol  and  that  this  view  was  only  finally  discredited  by  a 
study  of  the  pure  crystalline  pigment. 

Cotte  3  recently  carried  out  an  investigation  in  which  he  sought 
and  claims  to  have  shown  that  the  lipochromes,  both  animal  and 
vegetable  are  intimately  associated  with  cholesterol.  Cotte's  results 
have  been  thoroughly  disproved  by  Henze.4 

Since  the  early  work  of  Pouchet  5  and  Maly  6  who  distinguished 
between  yellow  and  red  crustacean  lipochromes  many  investigators 
have  classified  the  lipochromes  according  to  their  red  or  yellow  color, 
Newbigin 7  in  a  recent  investigation  of  the  pigments  of  the  skin, 
muscle  and  ovaries  of  the  salmon,  reports  that  he  found  two  pigments 
present,  a  red  and  a  yellow,  which  he  claims  he  was  able  to  separate 
from  each  other.  Newbigin  concluded  from  the  color  reactions  of 
the  pigments  that  the  red  pigment  was  a  true  lipochrome  while  the 
yellow  pigment  was  not. 

In  regard  to  the  yellow  pigment,  Newbigin  says  that,  "It  belongs 
to  a  group  of  pigments  that  are  apparently  exceedingly  widely  dis- 
tributed in  the  animal  kingdom,  but  which  have  been  little  investigated. 
They  have  been  commonly  confounded  with  the  lipochrome  pigments." 

He  extracted  the  pigment  from  the  bright  yellow  body  fat  of  a 
cow  and  found  it  to  have  properties  identical  with  the  yellow  pigment 

1.  Jour.  Physiol.  9,  p.  1  (1888). 

2.  Quart  Jour.  Micros.  Sc.  30,  p.  15  (1889). 

3.  Compt.  Rend.  Soc.  Biol.  55,  p.  812   (1903). 

4.  Zeit.  Physiol.  Chem.  41,  p.  109    (1904). 

5.  Jour.  d.  1'Anat.  de  la  Physiol.  1,  12,  10  (1876). 

6.  Sitz.  d.  k.  Akad.  d.  Wiss.  zu.  Wein.  83  (1831). 

7.  D.  Noel  Patton— Report  of  Inv.  on  Life  Hist,  of  Salmon  ( 1898 ) ,  Article  XT. 


328        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

of  the  salmon  with  the  exception  that  it  was  very  little  soluble  in 
methyl  alcohol,  but  dissolved  readily  in  ether. 

General  Properties  of  the  Lipochromes.  It  will  not  be  out  of  place 
to  give  a  brief  summary  here  of  the  general  characteristics  and  prop- 
erties of  the  lipochrome  pigments  as  found  up  to  this  time. 

Lipochromes 7  may  be  classed  as  salve-like,  yellow  or  red  or 
orange  colored  residues,  which  have  been  obtained  in  needles  or 
rhombic  plates,  where  they  have  been  crystallized.  They  are  soluble 
in  alcohol,  ether,  benzol,  petroleum  ether,  amyl  alcohol,  chloroform, 
carbon  bisulphide,  ethereal  oils  and  fats  with  a  yellow  or  yellow- 
orange  color.  They  are  insoluble  in  cold  and  hot  water  and  alkalies 
and  dilute  acids,  but  are  soluble  in  alcoholic  alkaline  solutions  and 
are  unchanged  when  these  solvents  are  heated.  In  alcohol  or  other 
solvents  they  are  unstable,,  and  readily  bleach,  as  do  the  residues 
from  these  solutions.  The  bleach  product  is  unknown,  but  it  is  cer- 
tainly not  identical  with  cholesterol.  On  addition  of  concentrated 
H2  SO4  or  HNO3,  the  lipochromes  give  a  color  change  of  blue-green- 
violet  to  brown.  The  color  reactions  are  often  interfered  with  by  the 
presence  of  a  small  amount  of  foreign  substance.  The  lipochromes 
generally  give  a  blue-green  coloration  with  a  solution  of  iodine  in 
potassium  iodide.  Spectroscopically  the  lipochrome  solutions  show 
two  bands  and  sometimes  three  in  the  blue  part  of  the  spectrum, 
and  again  they  sometimes  show  no  bands  at  all. 

The  lipochromes  may  be  extracted  from  the  fresh  or  dried  tissues 
in  which  they  are  found,  by  organic  solvents,  best  by  hot  or  cold 
alcohol,  ether,  petroleum  ether,  carbon  bisulphide  or  chloroform,  the 
choice  of  the  solvent  resting  with  whether  some  foreign  pigment  is 
present.  When  fat  is  present,  the  pigment  may  be  heated  with  alcoholic 
alkali  which  will  not  saponify  the  lipochromes.  The  lipochromes  can 
be  extracted  from  the  soap  with  ether,  petroleum  ether,  or  chloroform, 
either  directly  or  after  acidifying,  or  the  lipochromes  can  be  salted  out 
of  their  alkaline  soap  solutions  with  sodium  chloride,  and  the  lipo- 
chromes obtained  by  extracting  the  precipitated  soap  with  alcohol  or 
ether. 

The  Lipochromes  of  Algae,  Fungi,  and  Bacteria.  While  a  wide  dis- 
tribution of  the  lipochromes  has  already  been  mentioned,  a  review 
of  their  literature  would  not  be  complete  without  mentioning  their 
distribution  in  algae,  fungi  and  bacteria. 

1.  Summarized  from  "Lipochromes"  by  Franz  Samuely.  Alderhalden's 
Biochemisches  Handlexikon  vol.  6,  and  Handbuch  der  Biochemischen  Arbeits- 
methoden,  vol.  2. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    32^ 

Hansen  l  first  showed  the  presence  of  lipochromes  in  algae  and 
Tammes  2  has  lately  shown  their  presence  in  a  large  number  of  these 
plants.  Zopf 3  has  investigated  the  lipochromes  of  fungi  and  especi- 
ally of  bacteria,  the  first  lipochrome-producing  bacteria  being  pointed 
out  by  him. 

The  Lipoehrome  or  Lutein  of  Egg  Yolk.  It  will  be  readily  agreed 
that  while  some  order  has  been  attained  in  classifying  the  widely  dis- 
tributed animal  pigments,  by  means  of  the  convenient  and  flexible 
classification  "lipochromes,"  our  knowledge  of  the  animal  pigments  is 
far  from  being  satisfactory  when  compared  with  the  status  of  the 
orange  and  yellow  plant  pigments,  the  carotins  and  xanthophylls.  The 
science  of  animal  chromotology  should  accordingly  be  exceedingly 
grateful  for  the  recent  work  of  Willstatter  4  and  Escher,  on  the  lutein 
of  egg  yolk,  the  result  of  which  has  been  to  throw  new  light  upon 
the  constitution  of  the  lipochromes  of  the  higher  animals  and  upon 
their  relations  to  the  carotins  and  the  xanthophylls. 

The  main  pigment  of  the  yolk  of  hen's  eggs  was  isolated  in 
crystalline  form  by  these  investigators,  and  when  in  approximately 
pure  condition  showed  sufficiently  close  agreement  with  the  constitution 
of  xanthophyll  that  the  authors  claim  that  the  egg  lutein  on  account 
of  its  melting  point  (195-196°  C.  corrected)  is  a  true  isomer  of  the 
crystalline  xanthophyll  of  green  plants.  In  all  its  other  properties  in- 
cluding its  spectroscopic  absorption  bands,  the  egg  lutein  was  identical 
with  the  crystalline  plant  xanthophyll. 

It  is  worthy  of  note  also  that  during  the  isolation  of  lutein  a 
minor  constituent  was  noticed  which  gave  every  indication  of  being 
closely  related  to  carotin;  but  as  it  was  present  in  very  small  amount 
compared  with  the  xanthophyll  it  was  disregarded. 

In  concluding  the  review  of  this  investigation  it  will  be  important 
to  mention  that  the  authors  state  that  one  of  them,  i.  e.,  Escher,  is  at 
present  investigating  the  pigment  of  the  corpus  luteum  which  they  state 
has  been  found  to  belong  to  the  hydrocarbon  or  carotin  group  of 
pigments.5 

1.  Arbeit  Botan.  Inst  Wurzberg  3,  296  (1883). 

2.  Loc.  cit. 

3.     Ber.  Botan.  Gessel.  9,  27  (1891). 

4.  Zeit  Physiol.  Chem.  76,  pp.  214-225    (1912). 

5.  Note — Since  writing  the  above,  Dr.  Escher  has  published  his  investiga- 
tions which  show  that  the  corpus  luteum  pigment  is  in  every  respect  identical 
with  the  carotin  of  the  carrot  and  of  green  plants.  Zeit.  f.  Physiol.  Chem.  83, 
p.  198  (1913). 


330        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

The  Physiological   Relation   Between  Plant  and  Animal   Lipochromes. 

With  the  review  of  the  chemical  side  of  this  problem  complete, 
it  yet  remains  to  consider  what  has  been  shown  in  regard  to  relations 
other  than  chemical,  between  the  animal  and  plant  pigments  whose 
properties  are  so  nearly  related  and  in  many  cases  identical. 

The  literature  has  been  found  to  be  very  brief  on  this  point. 
Newbigin  1  gives  a  rather  extensive  consideration  of  this  subject  and 
attempts  to  explain  the  presence  of  the  red  and  yellow  pigments  found 
by  him  in  the  salmon  organism.  While  he  considered  the  most  obvious 
explanation  to  be  that  they  were  derived  directly  from  the  food,  he 
found  a  number  of  difficulties  in  the  way  of  the  acceptance  of  such 
an  explanation,  the  most  important  of  which  was  that  he  was  able 
to  show  the  presence  of  but  a  trace  of  only  the  yellow  pigment  in  the 
usual  food  of  the  salmon, 

As  to  the  possibility  of  transference  of  yellow  pigments  from 
one  organism  to  another,  Newbigin  points  out  what  he  believes  to  be 
some  evidence  apart  from  the  case  of  the  salmon.  He  says,  "Poulton  2 
has  shown  by  experiment  that  certain  caterpillars  derive  their  pigments 
from  their  food.  Again  it  is  not  uncommon  to  find  fat  of  sheep  and 
cows  dyed  a  deep  yellow  color.  According  to  some  authorities  this 
occurs  quite  sporadically  without  known  cause,  while  according  to 
others,  special  foods,  notably  maize,  are  the  important  agents."  New- 
bigin says  in  this  connection,  "I  have  examined  the  yellow  pigment  of 
maize,  and  compared  it  with  the  pigment  from  yellow  fat.  The  maize 
pigment  gives  the  lipochrome  reaction  faintly  with  H2  SO4  distinctly 
with  HNO3  while  the  fat  pigment  gives  no  lipochrome  reaction.  In 
other  respects,  in  tint,  solubility,  etc.,  the  pigments  closely  resemble 
each  other."  Newbigin  did  not  feel  warranted  to  conclude  from  these 
experiments  that  all  yellow  pigments  of  animals  are  derived  from 
their  food,  for  with  such  a  conclusion,  he  states,  "It  would  be  difficult 
to  understand  why  such  colored  fat  should  not  be  universal  in  herbivor- 
ous animals,  for  all  green  parts  of  plants  contain  also  a  certain  amount 
of  yellow  pigment." 

It  seemed  to  Newbigin,  however,  that  a  reasonable  explanation  for 
salmon,  domesticated  cattle  and  caterpillars  would  be  to  suppose  that 
when  they  ingest  a  moderate  amount  of  colored  fat  in  their  food, 
that  they  could  utilize  or  eliminate  the  pigment,  and  so  deposit  colorless 
fat  in  the  tissues ;  but  when  the  ingestion  of  colored  fat  is  in  excess 
of  the  actual  requirements  as  it  so  often  is,  especially  with  domes- 

1.  Loc.  cit. 

2.  Proc.  Roy.  Soc.  54,  p.  417;  Nat.  Sci.  8,  p.  98. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    331 

ticated  cattle,  an  elimination  or  utilization  of  the  pigmented  fat  is 
impossible  and  fat  colored  with  the  pigment  in  a  more  or  less  modified 
condition  is  thus  stored  up. 

There  is  abundant  proof  in  this  literature  aside  from  the  above 
speculations  that  animals  are  able  to  lay  up  fat  soluble  dyes  in  the 
organism  and  even  eliminate  them  in  the  milk.  Only  recently  Mendel 
and  Daniels  1  have  shown  that  Sudan  III  and  other  fat  soluble  dyes 
may  be  deposited  in  the  organism  in  adipose  tissue  and  bone  marrow 
when  introduced  into  the  organism  either  dissolved  in  fat  or  when 
fed  alone.  When  fed  with  fat  or  when  fat  was  present  in  the  alimen- 
tary tract  the  dyes  entered  the  organism  through  the  lymphatics  in 
solution  in  fat,  but  when  fat  was  absent,  through  the  portal  circulation 
dissolved  in  bile  in  which  they  are  nearly  all  soluble.  In  the  latter 
case  the  pigments  did  not  pass  beyond  the  liver  unless  fat  was  present 
to  transport  them,  in  which  case  only  they  were  subsequently  found 
in  the  blood.  When  fat  stained  food  was  fed  to  small  animals  (cats, 
rats,  guinea  pigs,  etc.)  in  lactation,  and  in  one  case  with  a  goat,  the 
dye  appeared  in  the  milk  shortly  after  the  first  feeding  of  the  dye. 
The  same  authors  feeding  fifteen  grams  of  Sudan  III  to  a  Holstein 
cow  for  three  successive  days  were  unable  to  detect  the  dye  in  the  milk. 
The  authors  also  made  the  interesting  observation  that  stained  fat 
does  not  traverse  the  placental  barrier;  the  blood  and  foetus  and 
fat  of  the  young  born  of  Sudan-stained  female  cats  and  rats  were  free 
from  the  dye. 

Gogitidse2  fed  hog  fat  (100  grams  per  day)  colored  with  Sudan 
III  to  a  bitch  and  after  two  days  found  the  dye  in  the  milk.  The 
body  fat  did  not  show  this  coloration  so  soon  and  then  not  so  clearly, 
in  fact  only  after  long  continued  feeding  of  the  stained  fat. 

Backhaus  3  studying  the  "Influence  of  Feed  and  Individuality  on 
the  Taste  and  Healthfulness  of  Milk,"  says  that  a  number  of  plants 
influence  the  color  of  milk  and  butter.  The  same  author  conducted 
several  pigment  feeding  experiments  with  cows.  Negative  results  were 
obtained  with  respect  to  the  milk  when  feeding  Fuchsin,  Bismark 
brown,  and  curcuma  powder,  although  the  feces  showed  the  pigments 
abundantly.  When  feeding  sodium  fluorescin  the  urine  was  affected 
but  not  the  milk.  When  feeding  methyl  violet,  however,  the  author 
was  able  to  show  that  this  pigment  was  carried  into  the  milk  fat  in 
a  reduced  condition  so  that  on  contact  with  the  air  and  with  the  aid 

1.  Jour.  Biol.  Chem.  13,  No.  1,  p.  72  (1912). 

2.  Zeit.  f.  Biol.  45,  353   (1904). 

3.  Berichte,  Landwirt.  Inst.  U.  Konigsberg  5  (1900). 


332        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

of  heat,  the  milk  fat  showed  an  intense  blue  coloration.     The  feces, 
however,  showed  the  pigment  in  an  unchanged  condition. 

Summary. 

The  foregoing  review  of  the  literature  has  shown  that  the  great 
number  of  pigments  that  exist  throughout  the  entire  plant  and  animal 
kingdoms  have  long  been  of  interest  from  a  scientific  standpoint.  The 
pigments  of  botanical  origin  have  been  thoroughly  and  exhaustively 
investigated.  This  is  especially  true  of  the  yellow  pigments  carotin 
and  xanthophylls,  and  their  chemical  constitution  and  properties  are 
now  established. 

The  yellow  and  orange  pigments  of  plants  were  at  first  classified 
in  one  group,  and  were  called  carotins,  the  name  being  derived  from 
the  pigment  of  the  carrot,  which  was  the  first  one  investigated.  A 
great  many  different  names  were  given  to  this  pigment  as  it  was 
independently  discovered  in  various  plants  but  the  identity  of  these 
pigments  with  the  carrot  pigment  has  now  been  established.  It  was 
eventually  discovered  that  the  carotins  are  always  accompanied,  especi- 
ally in  green  plants,  by  a  second  great  class  of  pigments  which  have 
been  called  xanthophylls,  whose  relation  to  carotin  has  but  recently 
been  established. 

As  the  work  on  plant  pigmentation  developed,  it  was  recognized 
that  the  general  properties  of  a  great  many  yellow  pigments  found  in 
animals  were  similar  to  the  so-called  carotins.  The  first  investigators 
classified  these  animal  pigments  under  the  name  lutein,  the  name 
being  derived  from  the  pigment  of  the  corpus  luteum,  which  was  the 
first  one  investigated.  The  name  lutein  was  extended  by  the  animal 
chromotologists  to  include  the  carotins  of  plants  and  its  related  pig- 
ments. Later,  when  the  animal  luteins  had  become  generally  recog- 
nized by  their  association  with  fat,  the  name  lutein  was  changed  to 
lipochrome  and  this  designation  was  also  extended  to  include  all  sim- 
ilar pigments  of  both  plants  and  animals. 

The  classification  of  the  plant  and  animal  pigments  which  is  at 
present  generally  accepted  is  to  restrict  the  names  carotin  and  xan- 
thophylls to  the  two  great  classes  of  yellow  plant  pigments,  and  to 
include  under  the  name  lutein  or  lipochrome  only  those  yellow  pig- 
ments which  are  considered  to  be  of  animal  origin. 

The  most  recent  work  in  the  field  of  animal  chromotology  has 
shown  that  the  luteins  can  also  be  subdivided  into  carotin  and  xan- 
thophyll  groups  depending  on  their  chemical  relation  to  the  carotin  or 
xanthophylls  of  plant  origin.  Accordingly  Schunck  l  has  shown  the 

1.     Loc.  cit. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    333 

spectroscopic  identity  of  the  egg  yolk  pigment  with  a  xanthophyll 
which  he  isolated  from  the  yellow  daffodil,  the  nasturtium,  and  green 
leaves.  Willstatter  and  Escher  l  have  confirmed  this  with  a  chemical 
analysis  of  the  egg  yolk  pigment,  showing  it  to  be  a  true  isomer 
of  the  crystalline  xanthophyll  of  green  plants;  they  have  called  it 
xanthophyll  B.  Escher  2  has  recently  published  his  investigation  show- 
ing that  the  pigment  of  the  corpus  luteum  is  identical  in  chemical 
composition  and  properties  with  the  carotin  of  green  plants. 

These  recent  discoveries  have  opened  the  way  for  an  extension 
of  such  investigations  to  other  yellow  animal  pigments  whose  isolation 
is  rendered  much  more  difficult  by  their  association  with  very  large 
quantities  of  fat  and  other  substances.  These  discoveries  have  also 
raised  the  question  whether  any  relation  other  than  chemical  exists 
between  the  yellow  animal  and  plant  pigments.  This  question  has 
never  been  investigated.  The  investigations  which  will  be  reported  in 
the  succeeding  papers  are  the  first  to  show  that  there  is  a  definite 
relation  other  than  chemical  between  the  yellow  plant  and  animal  pig- 
ments. 

1.  Loc.  cit. 

2.  Loc.  cit. 


334        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 


BIBLIOGRAPHY. 

1.  Arnaud:     Compt.  rend.  100,  p.  751  (1885);  Jour.  Pharm.  Chim. 

14;  p.  149  (1886)  ;  Compt.  rend.  102,  p.  1119  (1886)  ;  104,  p. 
1293  (1887). 

2.  Backhaus:     Bericht.     Landwirt.     Inst.  U.  Konigsberg,  5  (1900). 

3.  Berzelius:    Ann.  d.  Chem.  21,  p.  257  (1837). 

4.  Borodin:     Melanges  Biol.  tir.  d.     L'Acad.     Imp.  d.  St.  Petersb. 

11,  p.  512  (1883). 

5.  Bouchardat:     Schweizg.  Jour.  Chem.  58,  95  (1830). 

6.  Bougerel:    Ber.  Chem.  Gessel.  10,  p.  1173  (1877). 

7.  Capranica:    Arch.  f.  anat.  u.  Physiol.  p.  283  (1877). 

8.  Conrad:     Flora  25  (1872). 

9.  Cotte:  Compt.  rend.  Soc.  Biol.  55,  p.  812  (1903). 

10.  Czapek:    "Biochemie  der  Pflanzen"  vol.  i,  p.  172  et  seq. 

11.  Desmoliere:    Chem.  Centr.  2,  p.  1001  (1892). 

12.  Ehrung:    Botan.  Cent.  69,  p.  154  (1897). 

13.  Eiiler  and  Nordenson:    Zeit.  f.  Physiol.  Chem.  56,  p.  223  (1908). 

14.  Filhol:    Compt.  rend.  39,  pp.  9-184;  50,  pp.  545  and  1182. 

15.  Fremy:     Ann.  Sc.  Nat.  13,  p.  45   (1860);  Compt.  rend.  41,  p. 

189  (1865). 

16.  Gogitidse:    Zeit.  f.  Biol.  45,  p.  353  (1904). 

17.  Halliburton:    Jour.  Physiol.  7,  p.  324  (1886). 

18.  Hansen:      Sitz.    ber.    d.    phys.    Med.    Ges.    Wiirzberg    (1883); 

Arbeiten  d.  Botan.  Gessel.  Wiirzberg.  3,  p.  127  (1884)  ;  "Die 
Farbstoff  des  Chlorophylls"  (1889). 

19.  Hansen:     Arbeit,  botan.  Inst.  Wiirzberg  3,  p.  296  (1883). 

20.  Hartsen:    Arch.  Pharm.  207,  p.  166  (1875). 

21.  Henze:    Zeit.  Physiol.  Chem.  41,  p.  109  (1904). 

22.  Hilger:    Botan.  Centr.  57,  p.  335  (1894). 

23.  Husemann:    Lieb.    Ann.  117,  p.  200  (1860). 

24.  Immendorff:    Landwirt.    Jahrb.  18,  p.  507  (1889). 

25.  Kirchner:    Dissert.  Erlangen  (1892). 

26.  Kohl :    "Untersuch.  iiber  d.  Karotin,"  Leipsig  1902. 

27.  Kraus:    Flora,  p.  155  (1875). 

28.  Krukenberg:     Central  f.  d.  Med.  Wiss.   1879.     Vergl.   Physiol. 

Studien  I  Reihe,  II  Abth.  pp.  67-71;  III  Abth.  pp.  114-115; 
IV  Abth.  pp.  30-35;  V  Abth.  pp.  87-94;  II  Reihe  i  Abth.  pp. 
165-167;  III  Abth.  p.  135. 

29.  Krukenberg:     Sitz.  ber.  d.  Jen.  Gessel.  f.  Med.  1885. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT.    335 

30.  Kiihne:    Utersuch.  des.  Physiol.  Inst.  U.  Heidelberg  I,  4th  Heft 

p.  341   (1878);  Jour.  Physiol.  i,  p.  109  (1878). 

31.  Kiihne:     Untersuch.  des.  Physiol.  Inst.  U.  Heidelberg  4,  p.  169 

(1882). 

32.  Maly:    Sitz.    d.  k.  Akad.  d.  Wiss.  zu  Wein  83  (1881). 

33.  MacMunn:     Philos.  Trans.  Roy.  Soc.  177,  p.  243  (1886);  Jour. 

Physiol  9,  p.  I  (1888);  Quart.  Jour.  Micro.  Sc.  30,  p.  is 

(1889). 

34.  Mendel  and  Daniels:    Jour.  Biol.  Chem.  13,  No.  i,  p.  72  (1912), 

35.  Merejkowsky:    Bull.  d.  la  soc.  Zool.  d.  France,  1883. 

36.  Molisch:    Ber.  d.  d.  Botan.  Gessel.  14,  p.  18  (1896). 

37.  Montanari:     Le  Stazioni  Sperim.  Agr.  Ital.  37,  p.  909  (1904). 

38.  Monteverde :    Acta.  Horti.  Petropolatini  XIII.  Nr.  9,  pp.  123  and 

150  (1893). 

39.  Monteverde  and  Lyubimenko:     Bull.     Acad.     Imper.     Sc.     St. 

Petersb.  30,  p.  609  (1912). 

40.  Newbigin:    D.  Noel  Paton.     "Report  of  Investigations  on  Life 

History  of  the  Salmon."    1898,  Article  XV,  p.  159. 

41.  Pabst:    Arch.  Pharm.  230,  p.  108  (1892). 

42.  Passerini:    Compt.  rend.  100,  p.  875  (1885). 

43.  Piccolo  and  Lieben:    Giornals  d.  Scienze  Naturali  et  Economich. 

Palermo  2,  p.  258  (1866). 

44.  Pouchet:    Jour.  d.  L'Anat.  d.  la  Physiol.  12,  p.  10  (1876). 

45.  Poulton:     Proc.  Roy.  Soc.  54,  p.  417;  Nat.  Sci.  8,  p.  98. 

46.  Pringsheim:     Untersuch.  uber  das  chlorophylls  I  Abth.,  Berlin 

1874. 

47.  Schrotter:    Verb.  Zool.  Botan.  Gessel.  44,  p.  298  (1895). 

48.  Schuler:    Dissert.  Erlangen  1899. 

49.  Schunck:     Proc.  Roy.  Soc.  65  (1899)  ;  68  (1901),  72  (1903). 

50.  Schunck:     Proc.  Roy.  Sec.  44,  p.  449. 

51.  Schwalbe:      Hand.    d.    Ges.    Augenheilkunde    von    Graefe    u. 

Saemisch  I,  p.  414  (1874). 

52.  Sorby:    Proc.    Roy.  Soc.  21,  p.  456  (1875). 

53.  Stadeler:    Jour.  f.  Pract.  Chem.  100,  p.  149  (1867). 

54.  Tammes:    Flora,  p.  205  (1900). 

55.  Thudichum:    Proc.  Roy.  Soc.  17,  p.  253  (1869). 

56.  Tschirch:    Botan.  Zeitung  42,  p.  817  (1884). 

57.  Tschirch:     Ber.  der.  d.  Botan.  Gessel   14,  pt.  2,  p.  76    (1896) 

22,  p.  414  (1904). 

58.  Tswett:    Ber.  der.  d.  Botan.  Gessel.  24,  pp.  316  and  384  (1906)  ; 

29,  p.  630  (1911). 


336        MISSOURI  AGR.  EXP.  STA.  RESEARCH  BULLETIN  No.  9. 

59.  Wachenroder:      Dissertatio    de    Anthelminticis    Gottigen    1826; 

Geiger's  Magaz.  Pharm.  33,  p.  144  (1831)  ;  Berzelius  Jahres- 
ber.  12,  p.  277  (1833). 

60.  Wiesner:     Flora   (1874);  Sitz.  der  Wein.  Akad.  89,  part  i,  p. 

325. 

61.  Willstatter  and  Escher:    Zeit.  f.  Physiol.  Chem.  64,  p.  74  (1910)  ; 

76,  pp.  214-225  (1912). 

62.  Willstatter  and  Meig:    Ann.  d.  Chemie  355,  p.  i  (1907). 

63.  Wittich:    Arch.  f.  Path.  Anst.  27,  p.  573  (1863). 

64.  Wirth:    Dissert.    Erlangen  (1891). 

65.  Zeise:     Lieb.     Ann.  62,  p.  380  (1847);  Ann.  Chem.  Phys.   (3) 

20,  p.  125  (1847). 

66.  Zopf :    Ber.  der  d.  Botan.  Gessel.  9,  p.  27  (1891). 


CAROTIN— THE  PRINCIPAL  NATURAL  YELLOW  PIGMENT  OF 
MILK  FAT— JPART  II.* 


Chemical  and  Physiological  Relations  of  Pigments  of  Milk  Fat  to  the 
Carotin  and  Xanthophylls  of  Green  Plants. 


LEROY  S.  PALMER  AND  C.  H.  ECKLES 

For  a  number  of  years  the  great  variety  of  yellow  animal  pig- 
ments have  been  classified  under  the  general  name  lipochrome.  Recent 
investigations  by  Willstatter  and  Escher,1  and  by  Escher  2  have  shown, 
however,  that  some  of  these  pigments  are  in  reality  very  closely  related 
or  identical  with  the  carotin  or  xanthophyll  pigments  of  plants.  Will- 
statter and  Escher  have  analyzed  the  pure  lutein  of  egg  yolk  and 
found  it  to  be  isomeric  with  the  crystalline  xanthophyll  of  green 
plants;  and  Escher  has  identified  in  the  same  manner  the  lipochrome 
of  the  corpus  luteum  as  a  true  carotin.  The  isolation  of  both  pig- 
ments was  attended  with  great  difficulty.  In  the  case  of  the  egg  yolk 
pigment  the  yolk  of  6,000  hen  eggs  yielded  only  four  grams  of  crude 
crystalline  pigment,  while  in  the  case  of  the  corpus  luteum  pigment 
less  than  0.5  gram  of  crystals  were  obtained  from  10,000  cows'  ovaries. 

The  natural  yellow  pigment  of  butter  is  the  most  commonly  ob- 
served of  all  animaF  lipochromes.  It  is  also  more  important  from  a 
commercial  standpoint  than  any  other  animal  pigment;  the  public 
judges  the  richness  of  dairy  products  by  their  yellow  color,  and  de- 
mands that  butter  especially  shall  have  a  standard  shade  of  yellow. 
The  pigment  of  butter  fat,  however,  has  been  the  least  investigated 
of  all  animal  lipochromes.  Thudichum's 3  classical  investigation  in- 
cluded the  pigment  of  butter  fat  under  the  general  classification  lutein, 
which  he  proposed.  No  other  study  of  the  butter  fat  pigment  has 

1.  Zeit.  f.  Physiol.  Chem.  76,  pp.  214-225  (1912). 

2.  Zeit.  f.  Physiol.  Chem.  83,  p.  198  (1913). 

3.  Proc.  Roy.  Soc.  17,  p,  253  (1869). 


*  See  Res.  Bui.  No.  9,  p.  312,  for  statement   of  co-operation   with   U.   S. 
Dept.  of  Agriculture. 


(339) 


34°   MISSOURI  AGRICULTURAL  EXPERIMENT  STATION,  BULLETIN  NO.  IO 

been  reported.  It  is  usually  classified  in  the  current  text  books,1 
however,  according  to  Krukenberg's  classification  of  lipochromes. 

In  view  of  the  commercial  importance  attached  to  the  butter  fat 
pigment  and  especially  in  view  of  the  results  of  the  most  recent  inves- 
tigations in  this  field,  it  was  recognized  that  a  thorough  investigation 
of  this  pigment  would  be  of  great  value  both  from  a  scientific  as  well 
as  a  practical  standpoint. 

The  present  investigation  was  therefore  undertaken  for  the  pur- 
pose of  classifying  the  butter  fat  pigment  as  a  true  lipochrome  and 
also  with  respect  to  its  relation  to  the  carotin  and  xanthophylls  of 
green  plants.  It  was  also  the  purpose  of  the  investigation  here 
recorded,  to  gather  as  much  information  as  possible  relative  to  the 
influence  of  certain  factors  upon  the  color  of  butter,  among  which  may 
be  mentioned  the  character  of  the  ration  and  the  breed  of  the  cow. 


METHODS  OF    ISOLATION, 

The  statement  is  frequently  met  in  the  literature2  and  in  the  text 
books  and  works  8  on  oils  and  fats,  that  the  pigment  of  butter  or  butter 
fat  appears  in  the  unsaponifiable  extracts  along  with  cholesterol  and 
other  substances. 

A  number  of  methods  for  obtaining  the  unsaponifiable  matter  of 
butter  fat  are  available  and  several  were  tried.  The  method  finally 
adopted  for  the  isolation  of  the  crude  pigment  was  to  saponify  the 
butter  fat  with  a  twenty  per  cent  solution  of  alcoholic  potash,  using 
2,  c.  c.  for  each  gram  of  fat.  Saponification  was  allowed  to  continue 
for  one  half  to  one  hour  at  the  temperature  of  the  boiling  solution. 
The  soap  was  dissolved  in  three  volumes  of  distilled  water.  After 
cooling,  the  solution  was  shaken  with  an  equal  volume  of  pure  ether 
in  a  separatory  funnel.  The  extraction  was  repeated  with  a  fresh 
volume  of  ether  equal  to  one-half  the  volume  of  the  soap  solution. 
It  was  found  that  this  procedure  would  leave  the  soap  colorless,  if 
no  aldehyde  resins  had  formed  during  saponification  or  none  of  these 
colored  bodies  had  been  present  in  the  alcoholic  potash  previous  to  its 
addition  to  the  fat.  The  ether  extract  containing  the  pigment  and 
other  unsaponifiable  matter  was  now  freed  from  alkaline  soap,  by 
shaking  many  times  with  excess  water,  carefully  at  first  to  avoid 

1.  Such  as  Hammarsten,  "Text  Book  of  Physiological  Chemistry"    and 
Schaefer,  "Text  Book  of  Physiological  Chemistry",  etc. 

2.  Kirsten:  Zeit.  Nahr.  Genuss.  5,  p.  833  (1903). 

3.  Lewkowitsch:    "Oils,  Fats  and  Waxes."     Vol.  I,  p.  371,  (1909  Edition). 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK  FAT         34! 

emulsions,  and  more  vigorously  with  subsequent  washings.  When 
the  wash  water  no  longer  reacted  alkaline  toward  phenolphthalein,  the 
ether  was  either  dried  over  fused  CaCl2  or,  after  standing  several 
hours,  decanted  from  the  precipitated  moisture.  The  ether  was  then 
evaporated,  leaving  a  salve-like  residue  of  various  tints  of  yellow  to 
orange  to  red,  depending  upon  the  amount  of  fat  used  and  the  depth 
of  its  original  color. 

Although  the  methods  of  study  eventually  adopted  did  not  make 
the  procedure  necessary,  it  was  found  possible  to  completely  free 
the  pigment  from  its  chief  impurity,  i.  e.,  cholesterol,  by  means  of 
the- digitonin  method  of  Windaus x  for  the  quantitative  estimation 
of  cholesterol.  A  hot  one  per  cent  solution  of  digitonin  in  ninety 
per  cent  alcohol,  when  added  to  an  alcoholic  solution  of  the  un- 
saponifiable  residues  from  butter  fat,  completely  precipitated  the 
cholesterol  as  a  colorless  compound,  leaving  the  pigment  in  solution. 
This  solution  was  still  contaminated,  however,  with  traces  of  fat  and 
lecithin  decomposition  products. 

GENERAL   PROPERTIES  OF  THE   BUTTER   FAT   PIGMENT. 

The  unsaponifiable  residues  from  butter  fat,  either  crude  or  freed' 
from  cholesterol  by  digitonin,  were  readily  soluble  in  hot  alcohol  and 
in  ether,  chloroform,  petroleum  ether,  etc.  with  a  golden  yellow  color; 
and  in  carbon  bisulphide  with  a  color  which  varied,  according  to  the 
concentration,  from  a  red  orange  to  a  blood  red  color.  The  freshly 
prepared  crude,  and  also,  the  cholesterol-free  residues  usually  gave 
an  instantaneous  but  transient  purple  color  on  adding  a  drop  of  con- 
centrated sulphuric  acid,  a  light  green  color  quickly  changing  to  a 
greenish  blue  color  on  adding  a  drop  of  concentrated  nitric  acid,  and 
a  dark  blue  color  with  the  combined  acids.  These  color  reactions 
were  usually  shown  a  little  clearer  by  the  cholesterol  free  residues. 
Small  amounts  of  impurities  often  interfered  with  the  color  reactions ; 
in  fact  they  were  often  rendered  negative  by  some  slight  decompo- 
sition of  the  pigment  which  was  not  apparent  in  the  intensity  of  the 
color.  This  was  -due  to  the  fact  that  the  crude  samples  of  butter  fat 
pigment  were  very  unstable,  quickly  bleaching  in  the  air,  especially 
with  the  aid  of  heat  in  the  presence  of  a  little  water.  It  was  necessary 
therefore  to  take  great  care  to  have  the  ether  solutions  of  the  pigment 
as  free  as  possible  from  water  before  evaporation,  or  to  transfer  the 
pigment  to  some  solvent  such  as  petroleum  ether,  which  does  not 
absorb  water  so  readily. 

1.     Zeit.  f.  Physiol.  Chem.  65,  p.  110  (1909). 


342    MISSOURI  AGRICULTURAL  EXPERIMENT  STATION,  BULLETIN  NO.   IO 

In  addition  to  the  above  properties  it  was  found  that  all  fat-free, 
but  not  necessarily  cholesterol-free  solutions  of  the  pigment  showed 
spectroscopic  absorption  bands.  In  alcohol  two  sharp  bands  were 
exhibited  in  the  blue  part  of  the  spectrum;  and  in  carbon  bisulphide 
these  bands  were  nearly  always  accompanied  by  a  third  faint  band 
in  the  violet,  which  was  now  visible  on  account  of  the  general  shifting 
of  the  bands  toward  the  red  end  of  the  spectrum.  The  unstable  char- 
acter of  the  butterfat  pigment  required  that  the  isolation  be  carried 
out  as  rapidly  as  possible  in  order  to  preserve  all  the  characteristic 
properties  of  the  pigment.  This  was  especially  true  for  the  study  of 
the  absorption  spectra.  Fifteen  to  thirty  grams  of  fat  were  found 
to  yield  sufficient  pigment  for  a  spectroscopic  study.  The  use  of 
large  quantities  of  fat  (300  to  1,000  grams)  always  led  to  unsatis- 
factory results. 

The  general  properties  of  the  butter  fat  pigment  show  that  it  is 
to  be  classed  as  a  true  lipochrome.  The  chemical  relation  of  the  pig- 
ment to  the  carotin  and  xanthophylls  of  green  plants  remains  to 
be  shown. 

METHODS  OF  IDENTIFICATION. 

The  nature  of  the  substances  with  which  the  butter  fat  pigment 
is  associated  at  once  precluded  its  isolation  in  sufficient  quantity  to 
establish  its  chemical  composition  and  molecular  weight.  It  was 
therefore  necessary  to  adopt  other  methods  of  identification  which 
would  be  sufficiently  accurate  and  characteristic  that  the  final  results 
could  not  be  mistaken. 

The  methods  that  were  adopted  were,  (i)  a  study  of  the  spec- 
troscopic absorption  properties,  (2)  a  study  of  the  relative  solubility 
properties,  (3)  a  study  of  the  adsorption  properties  with  respect  to 
calcium  carbonate;  (4)  an  attempt  was  also  made  to  study  the  crys- 
talline form. 

Before  giving  the  results  of  our  studies,  some  discussion  will  be 
given  of  the  relative  solubility  and  adsorption  properties  of  carotin 
and  the  xanthophylls. 

RELATIVE   SOLUBILITY   OF   CAROTIN    AND   XANTHOPHYLLS. 

M.  Tswett  *  was  the  first  one  to  publish  a  comprehensive  state- 
ment in  regard  to  the  relative  solubility  of  the  plant  pigments  in 

1.     Ber.  der.  Deut.  Botan.  Gessel.  24,  pp.  316,  384  (1906). 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK  FAT         343 

different  solvents.  He  classified  the  solvents  into  three  groups  as 
follows  according  to  their  relation  to  the  leaf  pigments. 

"i.  Alcohols  (methyl,  ethyl,  amyl),  acetone,  acetaldehyde,  ether, 
chloroform:  These  solvents,  acting  on  freshly  cut  up  or  dried  leaves 
dissolve  out  all  pigments  equally  and  completely." 

"2.  Petroleum  ether  or  petroleum  benzin:  This  solvent,  acting 
on  fresh  leaves  finely  ground  with  sand  or  emery,  takes  on  a 
more  less  yellow  appearance,  which  is  especially  due  to  carotin, 
but  contains  also  other  pigments.  Dried  leaves  (at  a  low  temperature) 
likewise  give  up  their  carotin  to  this  solvent,  and  in  somewhat  purer 
condition." 

"3.  Benzol,  xylol,  toluol,  and  carbon  bisulphide:  These  solv- 
ents set  intermediately  between  the  first  two  groups." 

Willstatter  and  Mieg  J  a  little  later  approached  the  same  problem 
from  another  standpoint  and  showed  that  the  methods  used  by  Kraus  2 
and  Sorby  3  for  demonstrating  the  presence  of  more  than  one  pigment 
in  green  plants,  when  properly  applied  could  be  made  characteristic 
properties  of  carotin  and  xanthophylls.  Kraus  shook  his  alcoholic 
extracts  of  green  leaves  with  petroleum  ether  and  found  that  the 
green  pigment  went  into  the  petroleum  ether  leaving  the  alcoholic 
solution  yellow.  Sorby  shook  his  alcoholic  extracts  with  carbon 
bisulphide  and  found  that  the  latter  solvent  contained  the  green  pig- 
ment while  the  alcohol  was  left  yellow.  Wilstatter  and  Mieg,  apply- 
ing these  tests  to  the  isolated  carotin  and  xanthophyll  pigments  obtained 
the  following  results: 

"i.  If  methyl  alcohol  is  added  to  a  petroleum  ether  solution 
of  carotin  so  that  the  liquids  do  not  mix,  the  carotin  will  remain  for 
the  greatest  part  in  the  petroleum  ether  layer,  the  alcohol  layer  being 
only  slightly  colored.  If  a  trace  of  water  is  added,  the  methyl  alcohol 
layer  will  become  colorless.  The  same  phenomenon  occurs  with  ethyl 
alcohol,  and  one  can  start  with  an  alcohol  or  benzol  solution  and 
show  the  same  thing." 

"2.  If  carbon  bisulphide  is  added  to  an  alcoholic  carotin  solu- 
tion and  a  little  water  added,  the  carbon  bisulphide  will  separate  and 
will  quantitatively  contain  the  carotin." 

"3.  If  an  alcoholic  solution  of  the  xanthophylls  is  mixed  with 
petroleum  ether  and  the  liquids  separated  with  a  little  water,  by  far  the 
greatest  portion  of  the  xanthophylls  will  be  found  in  the  alcohol  layer." 

1.  Ann.  der.  Chemie  355  p.  8  (1907). 

2.  Flora,  p.  155  (1875). 

3.  Proc.  Roy.  Soc.  21,  p.  456  (1875). 


344   MISSOURI   AGRICULTURAL   EXP.   STA._,   RESEARCH   BULLETIN   NO.    IO 

"4.  If  an  alcoholic  solution  of  xanthophylls  is  mixed  with  carbon 
bisulphide  and  the  solvents  separated  with  water,  the  xanthophylls  will 
be  distributed  between  both  layers." 

Schunck,1  using  Sorby's  method,  showed  that  if  alcoholic  solu- 
tions of  xanthophylls  are  repeatedly  shaken  with  carbon  bisulphide 
all  the  xanthophylls  with  spectroscopic  absorption  properties  can  be 
extracted. 

Tswett2  studying  this  question  again  in  1911  states  that,  "Accord- 
ing to  a  well  known  rule,  organic  compounds  are  best  soluble  in  solv- 
ents of  similar  composition,"  and  concludes  that  carotin,  a  hydrocarbon, 
is  therefore  much  more  readily  soluble  in  the  hydrocarbons  of  the 
aliphatic  and  cyclic  group  than  in  alcohols,  a  point  well  illustrated 
by  the  above  experiments  of  Willstatter  and  Mieg.  Continuing,  Tswett 
states,  "If  one  therefore  shakes  an  eighty  to  ninety  per  cent  alcoholic 
solution  of  carotin  with  petroleum  ether,  the  pigment  goes  almost 
completely  into  the  petroleum  ether  layer."  "A  pigment  which  in 
the  above  mentioned  two  phase  system  occupies  the  lower  alcoholic 
layer,  is  therefore  not  a  carotin."  It  may  be  added  in  the  light  of 
Willstatter  and  Mieg's  investigation  that  if  the  original  solution  before 
differentiation  was  a  mixture  of  carotin  and  xanthophylls,  the  lower 
alcoholic  layer  will  contain  the  xanthophylls. 

ADSORPTION  PROPERTIES  OF  CAROTIN   AND  XANTHOPHYLLS. 

Considering  now  the  so-called  adsorption  properties  of  the  pig- 
ments, we  find  that  this  striking  characteristic  was  discovered  and 
elaborated  by  Tswett.3  This  investigator  found  that  by  shaking  a 
perfectly  anhydrous  petroleum  ether  or  carbon  bisulphide  solution  of 
the  mixed  pigments  of  green  plants  with  an  excess  of  dry  calcium 
carbonate,  Inulin  or  Saccharose,  all  the  pigments  will  be  completely 
adsorbed  by  the  material  with  the  exception  of  the  carotin,  which  can 
be  readily  washed  out  of  the  material  with  the  free  solvent.  In  the 
case  of  petroleum  ether  solutions  the  green  colored  mass  can  now 
be  completely  freed  from  all  its  pigments  by  means  of  petroleum  ether 
containing  ten  per  cent  absolute  alcohol.  If  the  resulting  solution 
is  now  shaken  with  eighty  per  cent  alcohol  the  petroleum  ether  layer 
will  contain  the  chlorophyll  pigments  and  the  alcohol  layer  the  xan- 
thophylls. "If  to  the  petroleum  ether  solution  of  the  mixed  pigments 

1.  Proc.  Roy.  72  (1903). 

2.  Ber.  der.  Deut.  Botan.  Gessel.  29,  p.  630  (1911). 

3.  Ber.  der.  Deut.  Botan.  Gessel.  24,  p.  316  and  384  (1906). 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         345 


is  added  adsorption  material  only  sufficient  to  destroy  the  fluorescence, 
both  the  carotin  and  xanthophylls  remain  in  solution,"  and  can  be 
separated  by  means  of  a  differentiation  between  the  petroleum  ether 
and  eighty  per  cent  alcohol,  or,  "By  treating  the  solution  with  more 
adsorption  material,  after  pouring  it  away  from  the  first,  and  the 
xanthophylls  then  freed  from  combination  with  the  adsorption  material 
by  means  of  alcoholic  petroleum  ether/'  In  addition  to  the  above, 
Tswett  made  the  interesting  discovery  that  the  pigments  which  are 
adsorbed  by  the  various  materials  suggested,  can  to  a  certain  extent 
displace  one  another  in  the  adsorbing  material.  As  an  example  one 
finds  that,  "If  a  petroleum  ether  solution  of  the  mixed  pigments  is 
filtered  through  a  column  of  adsorption  material  (such  as  CaCO3 
packed  tight  in  a  glass  tube)  the  pigments  will  be  separated  from  one 
another  from  top  to  bottom  in  differently  colored  zones,  proportion- 
ately to  their  degree  of  adsorption."  This 
separation  will  be  complete  if  a  stream  of 
pure  solvent  is  put  through  the  column 
after  the  pigment  has  been  adsorbed  in 
the  upper  part  of  the  column.  As  stated 
by  Tswett,  "Like  the  rays  of  light  in  the 
spectrum,  so  the  different  components  of 
a  pigment  mixture  are  actually  separated 
in  the  CaCO3  column,  and  may  thus  be 
qualitatively  estimated."  Tswett  calls 
such  an  experiment  a  "chromotogramm." 
He  found  carbon  bisulphide  to  be  one  of 
the  most  useful  solvents  for  a  chromoto- 
graphic  analysis,  on  account  of  the  bril- 
liant color  which  all  pigments  show  in 
this  solvent. 

In  describing  the  technique  for  the 
chromotographic  analysis,  the  author 
mentions  the  following  essential  points. 
A  very  finely  divided  material  with  not 
too  strong  adsorption  properties  should 
be  used  for  the  adsorbator.  (CaCO3  was 
found  to  answer  these  qualifications 
best.)  A  glass  tube  is  now  prepared  10  to 
20  m.  m.  in  diameter  and  15  to  20  c.  m. 
long,  one  end  of  which  is  drawn  out  to 

a  narrow  diameter,  at  which  end  the  opening  is  fused  in  a  little  to 
form  a  base  for  the  deposition  of  the  adsorbing  material.     A  small 


FIGURE  I. 


346    MISSOURI  AGRICULTURAL   EXP.   STA.,  RESEARCH   BULLETIN   NO.    IO 

piece  of  cotton  is  placed  in  the  small  end  of  the  tube  and  the  per- 
fectly dry  CaCO3  poured  in  and  firmly  tamped  down  to  a  homogenous 
texture.  The  chromotographic  apparatus  may  now  be  arranged  accord- 
ing to  Figure  I,  and  the  filter  flask  attached  to  the  suction  pump.  The 
CaCO3  is  now  moistened  with  a  little  of  the  solvent  to  be  used,  (this 
is  very  necessary)  and  a  certain  amount  of  the  liquid  which  is  to  be 
studied  poured  on  the  CaCO3.  A  stream  of  pure  solvent  is  subse- 
quently established  and  the  different  adsorption  zones  will  then  spread 
out  and  reach  their  definite  maximum  differentiation.  All  unadsorbed 
substances  will  be  completely  washed  away  and,  "Substances  which 
form  truly  dissociable  adsorption  compounds  with  the  CaCO3  pass 
slowly,  'ringwise'  through  and  can  be  taken  up  each  by  itself  at  the 
mouth  of  the  tube." 


Carotin  and  Xanthophyll  of  Green   Plants. 

After  selecting  the  methods  of  differentiation  and  characteriza- 
tion to  be  applied  to  the  milk  fat  pigment,  it  was  considered  desirable 
to  ascertain  whether  they  were  sufficiently  characteristic  for  a  com- 
plete identification,  should  the  milk  fat  pigment  be  found  to  be  either 
a  carotin  or  a  xanthophyll.  The  following  experiment  was  accord- 
ingly carried  out. 

About  fifteen  grams  of  air  dry  and  finely  divided  alfalfa  hay 
which  had  a  deep  green  color,  was  let  stand  for  several  days,  with 
shaking,  under  pure  carbon  bisulphide.  The  resulting  deep  olive  brown 
fluid  was  concentrated  to  about  25  c.  c.  at  a  low  temperature.  A 
glass  tube  about  eight  inches  long  and  one-half  inch  in  diameter, 
the  last  two  inches  of  which  were  drawn  out  to  a  small  opening, 
was  now  filled  and  packed  with  pure  CaCO3,  which  had  been  pre- 
viously dried  for  two  hours  at  150°  C.  The  CaCO3  was  tamped 
in  a  small  portion  at  a  time  by  means  of  a  small  cotton  wad  and  a 
heavy  glass  rod.  The  small  end  of  the  tube  was  now  inserted  through 
a  one  hole  rubber  stopper  and  fitted  tightly  into  a  side  neck  test  tube. 
The  apparatus  was  then  attached  to  a  suction  pump.  A  stream  of  pure 
carbon  bisulphide  was  passed  through  the  column.  When  the  CaCO3 
had  become  thoroughly  moistened,  2  to  3  c.  c.  of  the  alfalfa  extract  was 
poured  into  the  top  of  the  column,  vigorous  suction  being  maintained 
all  the  time.  When  the  extract  had  passed  entirely  into  the  CaCO3 
and  occupied  about  one  inch  of  the  column,  a  stream  of  pure  carbon 
bisulphide  was  run  through.  As  the  pigment  passed  through  it  dif- 
ferentiated itself  into  a  number  of  green  and  yellow  zones,  the  least 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK   FAT         347 


adsorbed  pigment  at  the  bottom  of  the  differentiation  having  a  rose 
color.  The  stream  of  carbon  bisulphide  was  continued  until  the  rose 
colored  solution  began  to  drop  from  the  lower  end  of  the  tube.  The 
appearance  of  the  column  at  this  time  is  shown  in  Figure  II.  The 
stream  of  carbon  bisulphide  was  now  continued  until  the  rose  colored 
zone  had  entirely  passed  through. 

According  to  Tswett  this  zone  contained  only  carotin.  The  beau- 
tiful rose  or  red  orange  solution  that  was  obtained  was  studied  as 
follows :  It  was  first  examined  in  the  spectroscope  where  it  showed  two 
distinct  bands,  and  a  faint  third  one.  (See  Table  i.)  The  carbon  bisul- 
phide solution  was  then  evaporated.  The 
residue  was  on  orange  yellow  solid.  A 
portion  of  it  gave  a  beautiful  blue  colora- 
tion with  concentrated  H2SO4. 

The  remainder  of  the  residue  was 
dissolved  in  hot  ninety-five  per  cent  alco- 
hol, and  the  phystosterol  which  precipi- 
tated out  on  cooling  filtered  off.  The  fil- 
trate was  divided  into  two  portions,  pe- 
troleum ether  (b.  p.  30-50°  C)  being 
added  to  one  and  pure  carbon  bisulphide 
to  the  other.  On  separation  of  the  re- 
spective solvents  with  a  little  water,  the 
pigment  was  found  quantitatively  in  the 
petroleum  ether  and  carbon  bisulphide 
layers  respectively.  The  pigment  was 
again  put  into  alcohol,  and  in  this  solv- 
ent showed  two  strong  absorption  bands 
and  end  absorption.  (See  Table  i.) 

Potassium  hydroxide  was  now  added 
to  the  alcoholic  solution  and  the  solution 
boiled  for  several  hours.     The  alkaline 
solution  was  diluted  with  three  volumes 
of  distilled  water  and  extracted  with  an 
equal  volume  of  ether.    The  ether  com- 
pletely and  readily  extracted  the  pigment,         FIGURE  II. 
showing  it  to  be  unsaponifiable.   The  golden  yellow  ether  solution  was 
washed  free  from  alkali  with  water  and  evaporated  to  dryness.  The 
residue  was  taken  up  with  alcohol.    This  solution  showed  only  two 
bands.    (See  Table  i.) 

The   alcoholic   solution   was   now   tested   again   for   its   relative 
solubility  toward  petroleum  ether  (b.  p.  30-50°  C)  and  carbon  bisul- 

3 


Colorless. 


Zone  I:   Yellow. 


Colorless. 

/  Zone  II: 
I  Yellow. 


Colorless. 

Green  Zone. 

/Zone  III: 

\  Yellow. 
Colorless. 

/  Zone  IV: 

\  Orange- Yellow. 

f  Greenish- 

1  Brown  Zone. 


Zone  V:  Rose. 


Cotton  Ping. 


348   MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 

phide.  The  pigment  was  again  found  quantitatively  in  the  petroleum 
ether  and  carbon  bisulphide.  A  portion  of  pigment  was  again  put  into 
alcohol  and  the  solution  made  strongly  alkaline  with  solid  sodium 
hydroxide.  On  addition  of  much  sodium  chloride  to  this  solution 
the  pigment  was  not  precipitated.  The  remainder  of  the  pigment 
was  now  dissolved  in  carbon  bisulphide,  after  evaporation  of  the  solu- 
tion (petroleum  ether),  and  the  carbon  bisulphide  solution  filtered 
through  the  CaCO3  column  again.  It  passed  through  unadsorbed 
as  a  rose  colored  zone. 

TABLE  1.     ABSORPTION  (a)  BANDS  OF  ALFALFA  CAROTIN 


In  CS2 

In  C2H6OH 

In  C2H6OH 
(after  ^ 
saponification) 

Band  I 
Band  II 
Band  III 

225-245 
259-279 
300-320 

256-275 
300-320 
345-.  .  . 

256-277 
303-320 

(a)  Note:  It  should  be  noted  that  all  spectroscopic  measurements,  both 
this  and  subsequent  ones,  were  made  according  to  an  arbitrary  scale  which  was 
attached  to  the  spectrometer.  This  scale  was  always  set  to  a  fixed  standard  be- 
fore studying  each  pigment,  the  standard  being  produced  by  a  sodium  flame 
with  the  spectrometer  slit  closed  to  furnish  the  narrowest  possible  line.  The 
spectrometer  was  equipped  with  a  crown  glass  prism  and  lenses  and  had  a  nar- 
row dispersion. 

The  study  of  the  alfalfa  carotin  showed  conclusively  that  its 
adsorption,  spectroscopic  and  solubility  properties  were  clear  and 
characteristic  and  were  unchanged  by  boiling  in  alcoholic  potash. 
It  was  also  shown  that  the  pigment  could  not  be  salted  out  of  its 
sodium  alcoholate  solution  with  common  salt  when  the  pigment  was 
free  from  fat.  If  the  solution  had  contained  much  soap  the  pigment 
would  in  all  probability  have  been  precipitated  with  the  soap  in 
the  salting  out  process.  The  object  of  the  test  was  to  see  whether 
this  was  or  was  not  a  characteristic  test  for  a  comparatively  pure  car- 
otin. Newbigin 1  claimed  to  have  found  a  true  lipochrome  which 
could  be  salted  out  of  its  alkaline  solution. 


1.     D.  Ndel  Paton.    "Investigations  on  Life  History  of  the  Salmon."    1898,. 
Art.  XV.  p.  159. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK   FAT         349 

After  studying  the  carotin  pigment,  more  carbon  bisulphide  was 
run  through  the  column  shown  in  Figure  II.  until  the  lower  green  zone 
was  washed  out.  This  was  removed  from  the  test  tube  and  the  stream 
of  carbon  bisulphate  continued  until  the  two  least  adsorbed  orange-yel- 
low zones  had  been  washed  out.  The  carbon  bisulphide  solution  of  these 
pigments  had  a  golden  yellow  color  with  a  faint  green  tinge.  Before 
the  spectroscope  the  solution  showed  two  chlorophyll  bands  in  the 
red,  and  three  very  clear  bands  in  the  blue.  (See  Table  2.) 

The  carbon  bisulphide  was  evaporated  off  and  the  residue  dis- 
solved in  hot  eighty  per  cent  alcohol,  the  phytosterol  which  precipi- 
tated on  cooling  being  filtered  off.  The  alcoholic  filtrate  was  now 
extracted  with  petroleum  ether  (b.  p.  30-50°  C),  which  took  up  some 
green  color.  This  was  continued  until  no  more  green  was  extracted. 
The  golden  yellow  alcoholic  xanthophyll  solution  was  examined  in 
the  spectroscope.  No  chlorophyll  bands  were  visible  but  only  two 
strong  bands  in  the  blue  and  sharp  end  adsorption. 

The  alcoholic  solution  was  now  saponified  with  potassium  hydrox- 
ide and  the  diluted  soap  extracted  with  ether  in  the  usual  way.  The 
color  immediately  went  into  the  ether,  the  separation  being  complete 
with  one  extraction.  After  washing  free  from  alkali  the  ether  was 
evaporated.  A  portion  of  this  residue  as  well  as  a  portion  of  the  res- 
idue of  the  solution  before  saponification,  showed  a  beautiful  green- 
ish blue  coloration  with  concentrated  H2SO4.  The  remainder  when 
dissolved  in  alcohol,  gave  a  golden  yellow  solution  showing  two  bands 
and  end  absorption.  (See  Table  2.) 

TABLE  2.     ABSORPTION  BANDS  OF  ALFALFA  XANTHOPHYLLS 


InCS2 

In  C2H6OH 

In  C2H6OH 
(after 
saponification) 

Band  I 
Band  II 
Band  III 

232-250 
273-293 
312-330 

265-282 
306-326 
357-.  .. 

265-286 
306-326 
355-.  .  . 

A  little  HC1  added  to  a  portion  of  the  alcoholic  solution  gave  no 
blue  coloration.  Solubility  tests  on  the  saponified  pigment  showed 
that  petroleum  ether  extracted  no  color  from  an  alcoholic  solution  on 
dilution  with  a  little  water,  while  carbon  bisulphide  extracted  about  an 
equal  portion. 


35O   MISSOURI  AGRICULTURAL   EXP.   STAV   RESEARCH   BULLETIN    NO.    IO 

These  tests  showed  conclusively  that  this  pigment  belongs  to  the 
oc  group  of  xanthophylls ;  and  that  its  adsorption,  solubility  and 
spectroscopic  properties  which  are  characteristically  different  from 
those  of  carotin,  are  unaltered  by  treatment  with  alcoholic  potash. 

The  remaining  xanthophyll  pigment  was  so  firmly  held  in  com- 
bination with  the  CaCO3  that  carbon  bisulphide  would  not  wash  it 
out.  A  stream  of  ten  per  cent  alcoholic  petroleum  ether  was  there- 
fore run  through  the  column,  washing  out  all  the  remaining  pigments. 
The  resulting  solution  showed  only  faint  absorption  bands,  indicat- 
ing that  the  pigments  noted  above,  i.  e.  carotin  and  xanthophyll  oc, 
are  the  principal  yellow  pigments  of  the  alfalfa  hay. 

IDENTIFICATION  OF  THE  PIGMENT  OF  BUTTER  FAT. 

With  the  properties  of  carotin  and  xanthophylls  well  established, 
attention  was  next  directed  to  the  butter  fat  pigment.  The  follow- 
ing experiments  were  carried  out,  the  results  of  which  are  very 
striking. 

Experiment  i. 

Fifteen  grams  of  very  yellow  butter  fat  from  a  Jersey  cow  who 
was  on  fresh,  green,  fall  grass  was  saponified  in  the  usual  way  with 
alcoholic  potash,  taking  great  care  to  avoid  the  presence  or  formation 
of  the  colored  alcohol  decomposition  products,  the  aldehyde  resins. 
After  dilution  the  soap  was  extracted  with  ether.  The  ethereal  extract 
was  washed  free  from  alkali  with  distilled  water  and  evaporated  into 
ninety-five  per  cent  alcohol.  An  equal  volume  (100  c.  c.)  of  petroleum 
ether  (b.  p.  30-50°  C.)  was  now  added  to  the  alcoholic  solution  and 
just  enough  water  to  cause  a  separation  of  the  alcohol  and  petroleum 
ether.  The  golden  yellow  petroleum  ether  layer  which  resulted,  con- 
tained practically  all  the  color.  The  alcohol  layer  was  drawn  off 
and  extracted  with  fresh  volumes  of  petroleum  ether  until  only  a 
trace  of  color  went  into  the  petroleum  ether  layer.  All  the  petroleum 
ether  extracts  were  then  combined  and  extracted  with  eighty  per 
cent  alcohol.  A  mere  trace  of  color  went  into  the  alcohol.  The 
alcohol  solutions  were  combined. 

The  Petroleum  ether  solution — This  contained  by  far  the  greatest 
part  of  the  total  pigment.  The  solution  was  evaporated  quickly  at 
a  temperature  below  50°  C.,  leaving  a  red  oily  residue  which  instantly 
dissolved  in  carbon  bisulphide  with  a  deep  red  orange  color.  After 
adjusting  the  concentration  for  the  10  m.  m.  cell,  so  that  the  bands 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         351 


were  all  plainly  visible,  this  solution  showed  three  distinct  absorption 
bands.  (See  Table  3.) 

The  carbon  bisulphide  solution  was  evaporated  to  dryness  at  the 
lowest  possible  temperature,  the  residue  taken  up  in  50  c.  c.  of  hot 
ninety-five  per  cent  alcohol  and  hot  one  per  cent  digitonin  in  ninety 
per  cent  alcohol  added  until  no  more  precipitate  came  down.  The 
digitonin-cholesteride  was  filtered  off,  the  filtrate  evaporated  to  dry- 
ness,  and  the  residue  dissolved  once  more  in  carbon  bisulphide.  The 
solution  still  showed  the  three  bands.  On  evaporation  it  left  a  golden 
yellow  oil  which  solidified  on  cooling  to  a  reddish  yellow  salve.  Con- 
centrated H2  SO4  added  to  the  residue  gave  a  blue  green  color  which 
slowly  changed  to  a  purple  color. 

The  alcoholic  solution. — This  was  evaporated  to  dryness.  When 
very  concentrated  it  showed  a  little  color,  and  the  carbon  bisulphide 
solution  of  the  residue  had  a  light  orange  color  when  it  had  a  volume 
of  i %  c.  c.  When  viewed  in  a  25  m.  m.  cell  this  solution  showed 
three  absorption  bands.  (See  Table  3.) 

TABLE  3.     ABSORPTION  BANDS  OF  BUTTERFAT  PIGMENTS. 


Petroleum  ether  soluble  pigment 
In  CS2  solution 

Alcohol  soluble 
pigment  in 
CS2  solution 

Crude 

Free  from 
Cholesterol 

Band  I 
Band  II 
Band  III 

222-240* 
257-276 
299-319 

224-242 
260-277 
301-316 

229-247 
269-288 
309-328 

The  striking  results  of  this  experiment  were  the  remarkable 
similarity  of  the  solubility  and  spectroscopic  properties  of  the  main 
butter  fat  pigment  to  carotin,  and  the  indications  of  a  secondary  minor 
constituent  of  the  butter  fat  pigment,  whose  solubility  and  spectro- 
scopic properties  were  strikingly  similar  to  xanthophyll. 

It  at  once  became  evident  that  should  these  observations  be  con- 
firmed, it  would  be  not  only  profitable,  but  essential  to  ascertain 
whether  the  presence  of  secondary  xanthophyll-like  pigments  is  nor- 
mal to  butter  fat  under  other  conditions  of  coloration,  such  as  in 
light  colored  butter  fat,  colostrum  butter  fat  and  other  conditions. 

That  the  presence  of  a  secondary  pigment  in  the  fat  under  inves- 
tigation was  confirmed  and  its  character  more  clearly  identified  is 
shown  by  the  following  experiment. 


352    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN    NO.    IO 

Experiment  2. 

Fifteen  grams  of  the  fat  was  treated  as  in  Experiment  i.  In  ad- 
dition the  carbon  bisulphide  solution  of  the  unsaponifiable  ether  extract 
was  filtered  through  a  column  of  CaCO3  in  a  manner  identical  with  the 
chromotographic  experiment  with  alfalfa  hay.  As  far  as  could  be  de- 
tected with  the  eye  all  the  pigment  passed  quite  rapidly  through  as  an 
unadsorbed  rose  colored  zone,  which  spread  out  considerably  in  its 
passage  through  the  column  but  showed  no  differentiation  into  zones. 
The  absorption  bands  of  the  filtered  pigment  were  identical  with  the 
bands  of  carotin. 

After  the  carbon  bisulphide  had  washed  out  all  the  pigment  and 
was  passing  through  colorless,  a  stream  of  petroleum  ether  contain- 
ing ten  per  cent  alcohol  was  run  through  the  column.  As  it  passed 
through  it  gathered  a  zone  of  yellow  color,  leaving  the  column  pure 
white.  This  pigment  was  collected  at  the  mouth  of  the  tube,  its  solu- 
tion evaporated,  and  the  residue  dissolved  in  carbon  bisulphide.  The 
light-orange  colored  solution  showed  two  strong  absorption  bands  and 
a  third  fainter  one. 

The  carbon  bisulphide  solution  of  the  main  pigment  was  now  evap- 
orated to  dryness  and  the  residue  dissolved  in  ninety-five  per  cent 
alcohol.  The  alcohol  was  diluted  with  water  to  an  eighty  to  ninety  per 
cent  solution  and  extracted  with  petroleum  ether  (b.  p.  30-50°  C).  The 
bulk  of  the  pigment  went  into  the  petroleum  ether  and  a  second 
extraction  with  fresh  petroleum  ether  took  out  still  more  pigment.  A 
third  extraction  with  fresh  petroleum  ether,  however,  left  the  alcohol 
layer  considerably  more  colored  than  the  petroleum  ether  layer.  The 
alcohol  layer  was  now  evaporated  and  the  residue  dissolved  in 
carbon  bisulphide,  giving  an  orange  yellow  solution  which  showed 
three  strong  absorption  bands.  There  seemed  to  be  three  or  four 
times  as  much  of  this  pigment  as  of  the  xanthophyll  which  had  been 
adsorbed  by  the  CaCO3  in  the  chromotogramm,  and  together  they 
probably  amounted  to  ten  per  cent  of  the  total  pigment. 

All  the  xanthophyll  pigments  were  now  combined  (they  were 
all  in  carbon  bisulphide  solution)  and  the  resulting  solution  analyzed  by 
means  of  a  chromotogramm.  As  the  orange-yellow  solution  was  washed 
through  the  column  by  a  stream  of  carbon  bisulphide  it  took  on 
the  appearance  as  shown  in  Figure  III.  Zones  two  and  three  were 
collected  together,  and  showed  three  absorption  bands.  (See  Table  4.) 
Their  solution  was  evaporated  and  the  residue  dissolved  in  petroleum 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT        353 


ether.  Only  a  part  of  the  residue  would  dissolve  readily,  and  the 
remainder  only  on  addition  of  a  little  absolute  alcohol.  Eighty  per  cent 
alcohol  extracted  a  large  part  of  the  color  from  the  alcoholic  petroleum 
ether  solution.  The  portion  which  remained  in  the  petroleum  ether  was 
transferred  to  carbon  bisulphide  in  which  it  showed  three  absorption 
bands.  (See  Table  4.)  The  portion  extracted  by  the  eighty  per 

cent  alcohol  was  also  transferred  to  car- 
bon bisulphide.  The  latter  solution  showed 
three  absorption  bands  but  the  third  was 
faint  and  was  not  measured.  (See  Ta- 
ble 4.) 

Alcoholic  solutions  of  both  portions 
of  pigment  showed  no  color  change  on 
the  addition  of  a  little  concentrated  HC1. 
There  was  also  no  effect  on  the  absorp- 
tion bands. 

Zone  i  of  the  chromotogramm  was  of 
a  pure  yellow  color.  It  was  completely 
adsorbed  by  the  CaCO3  with  respect  to 
CS2  and  was  evidently  the  same  pigment 
which  had  been  adsorbed  in  the  first  chro- 
motogramm of  the  combined  carotin  and 
xanthophyll-like  pigments.  A  stream  of 
alcoholic  petroleum  ether  readily  washed 
it  out  of  the  column  as  it  did  in  the  first 
chromotogramm.  In  carbon  bisulphide  so- 
lution the  pigment  had  a  light  orange 
color,  and  showed  two  brilliant  absorp- 
tion bands  and  a  third  fainter  one.  (See 
Table  4.) 

The  pigment  showed  the  three  bands 
in  alcoholic  solution  as  well  as  in  car- 
bon bisulphide.  When  in  alcohol,  it  gave 
no  color  reaction  with  a  little  concentrated 

HC1,  and  there  was  also  no  immediate  effect  upon  either  the  intensity 
or  position  of  the  absorption  bands.  In  the  solid  state  this  pigment 
gave  a  transient  greenish-blue  color  with  concentrated  H2SO4. 


Colorless. 


Zone  I: 
Yellow. 


Colorless. 


Zone  II: 
Orange, 
shading 
from  light 
to  dark. 
Zone  III: 
Deep 
Orange. 


Colorless. 


Cotton  Plug. 


FIGURE  III. 


354   MISSOURI  AGRICULTURAL  EXP.   STAV   RESEARCH   BULLETIN    NO.    IO 

For  the  sake  of  comparison  with  the  carotin  bands  of  the  alfalfa 
hay,  the  alcoholic  solution  of  the  main  butter  fat  pigment  was  exam- 
ined in  the  spectroscope.  The  results  are  given  in  Table  5. 


TABLE  4. 


ABSORPTION  BANDS  OF  BUTTER  FAT  XANTHOPHYLLS  SHOWN  IN 
FIGURE  III. 


Zone  I. 
CSa  solution 

Zones  II  &  III 
C2H6OH  solution 

Zones  II  &  III 

(P.  ether  sol. 
Part) 
InCS2 

(Alcohol 
sol.  Part) 
InCS2 

Band  I 
Band  II 
Band  III 

232-249 
271-288 
313-330 

263-280 
306-325 
355-.  .  . 

230-249 
268-289 
312-330 

231-250 
272-240 
Not  measured 

TABLE  5.     COMPARISON  OF  BANDS  OF  CAROTIN  OF  ALFALFA  AND  BUTTERFAT. 

(C2H6OH  SOLUTION). 


Alfalfa  Carotin 

Butterfat  Carotin 

Band  I 

256-275 

256-274 

Band  II 

300-320 

298-312 

Band  III 

345-.  .  . 

345-.  .  . 

The  result  of  this  experiment  was  not  only  to  confirm  the  re- 
markable similarity  of  the  main  butter  fat  pigment  of  this  partic- 
ular fat  to  the  carotin  of  alfalfa  hay,  but  also  to  confirm  the  presence 
of  a  secondary  constituent  practically  identical  with  the  xanthophylls. 
In  addition  it  was  found  that  these  xanthophylls,  like  the  xanthophylls 
of  alfalfa  hay,  could  be  chromotographically  separated  into  three 
constituents.  The  two  main  constituents  seemed  to  be  closely  related 
to  carotin  in  adsorption  properties  so  that  their  presence  could  not 
be  detected  in  the  presence  of  a  large  amount  of  carotin  until  first 
separated  from  the  main  pigment  with  the  aid  of  their  relatively 
greater  solubility  in  alcohol  with  respect  to  petroleum  ether.  The 
third  constituent  of  the  secondary  group  was  more  nearly  related 
to  a  true  xanthophyll  in  all  its  properties,  including  its  adsorption  by 
CaCO3.  When  classified  with  respect  to  the  action  of  their  alcoholic  so- 
lutions toward  a  little  concentrated  HCl  all  the  xanthophyll  pigments 
apparently  belong  to  the  group  which  Tswett  calls  oc  xanthophylls. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK   FAT         355 


STANDARDIZATION    OF    ABSORPTION    BANDS    OF    CAROTIN    AND 

XANTHOPHYLLS. 

Before  proceeding  further  it  may  be  well  to  make  a  few  state- 
ments in  regard  to  the  spectroscopic  properties  of  the  various  pig- 
ments which  have  been  under  consideration  and  which  are  still  to 
be  considered.  It  will  be  obvious  that  measurements  of  absorption 
bands  could  not  be  made  in  this  work  with  the  accuracy  attained 
by  Willstatter  and  his  collaborators  who  used  solutions  of  standard 
strength  and  a  spectroscope  of  great  exactness.  Small  differences 
in  measurement  among  xanthophylls  or  between  carotins  should  ac- 
cordingly be  disregarded.  It  was  merely  attempted  in  every  case 
to  secure  solutions  of  such  concentration  that  the  bands  were  of  as 
nearly  the  same  intensity  as  could  be  detected  with  the  eye  before 
making  measurements.  It  was  not  always  possible  to  use  the  same 
thickness  of  cell  to  secure  the  required  intensity  of  the  bands,  small 
amounts  of  pigment  naturally  requiring  a  greater  depth  of  solution 
than  large  amounts. 

In  order  to  have  standard  spectroscopic  properties  for  future 
comparison,  carotin  and  xanthophylls  were  extracted  from  the  car- 
rot, and  a  careful  study  made  of  the  spectroscopic  properties  of  each 
pigment. 

A  grated  carrot  was  boiled  in  water  for  about  two  hours,  the 
water  squeezed  out  of  the  pulp  and  the  pulp  dried  on  the  steam 
bath.  It  was  pulverized  and  extracted  with  ether  in  a  Soxhlet  extrac- 
tor until  colorless.  The  ether  was  evaporated  into  ninety-five  per 
cent  alcohol  at  a  low  temperature.  The  resulting  solution  was  diluted 
with  a  little  water  to  eighty  per  cent  and  the  pigment  carefully 
separated  between  petroleum  ether  (b.  p.  30-50°  C)  and  the  eighty 
per  cent  alcohol.  Both  portions  of  the  pigment  were  carefully  trans- 
ferred to  pure  carbon  bisulphide  and  the  solutions  adjusted  for  the 
10  m.  m.  cell  until  all  bands  were  distinct  and  as  nearly  as  possible 
of  equal  intensity.  With  the  spectrometer  set  at  the  sodium  line  stand- 
ard the  positions  of  the  absorption  bands  were  standardized.  The 
color  of  the  solutions  was  measured  also,  by  means  of  the  Lovibond 
tintometer.  (See  Figure  V.) 


356   MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN    NO.    IO 

This  data  is  given  in  Table  6. 

TABLE  6.     SPECTROSCOPIC  STANDARD  OF  CAROTIN  AND  XANTHOPHYLLIS.    (FROM 

THE  CARROT.) 


Pigment 

Layer 

Color                             Absorption  Bands 

Yellow 

Red 

Light 

Band  I 

Band  II 

Band  III 

InCS2 

10  m.m 
10  m.m 

26.0 
17.0 

5.5 
4.4 

1.0 
0.5 

225-242 
233-253 

261-278 
272-291 

301-319 
312-330 

Carotin 
Xanthophyll 

In  C2H6OH 

10  m.m 
10  m.m 



257-275 
263-280 

303-318 
305-325 

345-364 
355-.  .  . 

Carotin 
Xanthophyll 

It  will  be  noticed  that  the  relative  position  of  the  bands  of  car- 
otin and  xanthophylls  is  more  characteristic  in  carbon  bisulphide 
than  in  alcohol.  On  this  account,  nearly  all  subsequent  spectro- 
scopic  studies  were  made  in  carbon  bisulphide  solution. 

It  will  be  noticed  that  both  pigments  showed  three  bands.  The 
difference  in  the  color  of  the  carbon  bisulphide  solutions  of  the 
carotin  and  xanthophyll  pigments  when  showing  bands  of  equal  inten- 
sity is  also  especially  noteworthy.  To  the  eye,  the  carotin  solutions 
are  a  deep  red  orange  while  the  xanthophyll  solutions  are  a  much 
purer  orange  color.  It  is  worthy  of  mention  that  this  characteristic 
difference  was  so  noticeable  throughout  all  this  study,  that  in  all 
cases  the  color  of  the  carbon  bisulphide  solution  of  unknown  pig- 
ments was  recorded  in  connection  with  the  measuring  of  the  absorp- 
tion bands. 


CHARACTER  OF  THE   PIGMENTS   IN    DIFFERENT   BUTTER    FATS. 

It  was  stated  previous  to  the  confirmation  of  the  presence  of 
secondary  xanthophyll-like  pigments  in  the  particular  butter  fat 
studied,  that  if  the  presence  of  these  pigments  could  be  confirmed 
it  would  necessary  to  establish  such  a  fact  as  either  characteristic 
of  all  butter  fat  or  as  incidental  only  to  the  particular  fat  studied. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK   FAT         357 

A  wide  variety  of  fats  of  different  color,  from  different  breeds  of 
cows  and  produced  under  widely  different  conditions  of  feeding,  etc., 
were  available  for  this  study.  The  technique  of  these  experiments 
was  identical  with  that  used  for  the  high  colored  fat  from  the 
Jersey  cow  on  grass  recorded  in  Experiments  I  and  2  above. 

The  Pigments  of  Light  Colored  Fats. — The  character  of  the  pigment 
in  three  light  colored  fats  was  tested.  The  fats  represent  three 
subsequent  periods  in  a  feeding  experiment  of  a  pure  bred  Ayrshire 
cow  in  which  the  color  was  practically  eliminated  from  the  butter 
fat.  (See  Table  13  of  feeding  experiments  for  record  of  this  experi- 
ment.) The  color  of  the  fat  and  the  character  of  the  pigments 
are  shown  in  the  following  Table  7. 

TABLE  No.  7. 


Color  of  Fat 


Yellow  Red 


6.0 


2.5 


1.4 


1.0 


0.7 


0.5 


Character  of  Pigments. 


Petroleum  ether  quant,  extracted  cholesterol-free  pig- 
ment from  80  per  cent  alcohol. 

Solubility  test  showed  carotin  and  xanthophylls. 

Chromotogramm  of  xanthophylls  showed  adsorbed 
yellow  constituent  and  unadsorbed  orange  zone. 

Pigment  from  240  gm.  fat  had  color  of  35  yellow,  1.  0 
red.  Solubility  test  showed  both  carotin  and  xan- 
thophylls, as  did  also  absorption  bands. 


The  Pigments  of  Butter  Fat  After  Carrot  Feeding. — The  fat  tested 
was  taken  during  a  carrot  feeding  experiment  with  the  same  cow 
used  in  the  above  experiments.  This  feeding  experiment  directly 
followed  the  third  period  of  very  light  colored  fat.  The  color  of  the 
fat  was  28  yellow  and  1.4  red.  Solubility  tests  on  the  pigment 
showed  carotin  and  a  very  small  amount  of  xanthophyll.  The  petro- 
leum ether  soluble  part  of  the  pigment  gave  a  CS2  chromotogramm 
of  a  single  unadsorbed  rose  colored  zone.  The  filtered  pigment 
showed  three  well  defined  absorption  bands.  I,  225-245;  II,  263- 
283;  III,  301-320. 


358    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN    NO.    IO 


The  Pigments  of  the  Fat  from  Colostrum  Milk. — It  is  a  well  known 
fact  that  the  first  milk  drawn  after  parturition  always  has  a  high 
yellow  color.  It  is  not  generally  known,  however,  that  this  high 
color  is  usually  due  1  entirely  to  the  suspended  fat  globules.  We  have 
many  times  observed,  not  only  in  connection  with  this  study  but 
also  in  connection  with  numerous  studies  dealing  with  the  chemical 
composition  of  milk,  that  when  the  fat  is  entirely  removed  from 
colostrum  milk  the  skim  milk  has  the  appearance  of  ordinary  skim 
milk,  and  the  butter  and  the  rendered  fat  have  a  depth  of  color 
which  is  never  equaled  at  any  subsequent  stage  of  the  lactation  period. 
This  characteristic  of  colostrum  milk  is  common  to  all  breeds  of  cows, 
and  the  high  color  of  the  fat  continues  in  cows  of  all  breeds  for  a 
short  time  after  parturition  and  then  gradually  falls  off.  Table  8  gives 
the  color  of  the  milk  fat  of  several  cows  shortly  after  parturition  and 
again  a  week  or  two  later.  The  color  readings  are  the  Lovibond  tin- 
tometer readings  of  a  one-inch  layer  of  melted,  rendered  fat. 
TABLE  No.  8.  COLOR  OF  THE  FAT  OF  COLOSTRUM  MILK. 


Cow  No. 

Breed 

Roughage 

lCQ» 

Days  after 
Parturi- 
tion. 

Color 

Yellow 

Red 

Light 

301 

Ayrshire 

Alfalfa(a) 

4 

78.0 

3.5 

1.0 

301 

Ayrshire 

Alfalfa 

26 

71.0 

1.5 

0.5 

300 

Ayrshire 

Alfalfa 

4 

71.0 

3.5 

1.0 

300 

Ayrshire 

Alfalfa 

20 

68.0 

2.8 

1.0 

2 

Jersey 

Alfalfa 

13 

68.0 

2.6 

0.5 

2 

Jersey 

Alfalfa 

22 

57.0 

2.5 

0.5 

2 

Jersey 

Alfalfa 

2 

54.0 

4.3 

1.0 

2b 

Jersey 

Alfalfa 

20 

50.0 

2.5 

1.0 

20 

Jersey 

Alfalfa 

2 

47.0 

4.8 

1.0 

20 

Jersey 

Alfalfa 

25 

47.0 

2.0 

0.5 

206 

Holstein 

Alfalfa 

1 

50.0 

4.7 

0.3 

206 

Holstein 

Alfalfa 

5 

54.0 

2.0 

0.2 

(a)  The  Alfalfa  hay  was  rich  in  carotin  and  xanthophylls. 

(b)  Second  sample  taken  after  next  parturition. 

This   phenomenon   at   once   offered   the   interesting   problem   of 

the   relation   of   the   colostrum   pigment   to   the   pigment   of   normal 

butter.     A  close  study  was  accordingly  made  of  the  pigment  of  the 

fat  from  the  first  milk  drawn  after  parturition.     The  cow  selected 

1.     Colostrum  milk  is  occasionally  contaminated  with  blood. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         359 


for  study  was  a  pure  bred  Holstein.  The  fat  tested  had  a  very  high 
color;  a  one  inch  layer  gave  a  reading  of  64  yellow,  5.0  red  and 
i.o  light  in  the  Lovibond  tintometer. 

The  unsaponifiable  pigment  and  impurities  from  fifteen  grams 
of  the  fat  had  a  golden  yellow  color  in  ether  and  in  carbon  bisulphide 
a  blood  red  color.  This  solution  was  analysed  chromotographically. 
The  entire  pigment  passed  through  unadsorbed  as  a  red  orange  or 
rose  colored  zone,  leaving  no  adsorbed  zones  and  no  pigment  behind 
in  the  CaCO3  which  could  be  washed  out  with  ten  per  cent  alcoholic 
petroleum  ether.  The  filtered  solution  showed  two  strong  absorption 
bands  and  a  third  faint  one.  (See  Table  9.) 

The  pigment  was  now  analyzed  according  to  its  proportionate 
solubility  in  petroleum  ether  (b.  p.  30-50°  C)  and  eighty  per  cent 
alcohol,  and  was  thus  divided  into  two  portions,  a  major  portion  ex- 
tracted by  the  petroleum  ether  and  a  very  minor  portion  which  the  pe- 
troleum ether  would  not  extract  from  the  eighty  per  cent  alcohol. 

The  carotin-like  pigment  thus  obtained  was  freed  from  choles- 
terol by  the  digitonin  method  and  its  bands  again  measured  in  carbon 
bisulphide  solution.  (See  Table  9.) 

The  residue  from  this  solution  gave  a  beautiful  transient  blue 
color  with  concentrated  H2SO4  and  a  very  transient  blue-green  color 
with  concentrated  HNO3. 

The  eighty  per  cent  alcohol  soluble  pigment  showed  three  absorp- 
tion bands  in  carbon  bisulphide  solution,  the  first  two  being  a  little 
more  intense  than  the  third.  (See  Table  9.)  While  in  this  solution 
the  pigment  was  analyzed  by  means  of  a  chromotogramm  and  showed 
two  zones,  a  primary  little  adsorbed  zone  of  orange  color,  and  a  sec- 
ondary more  adsorbed  zone  of  yellow  color.  Hydrochloric  acid  gave 
no  color  reaction  with  the  alcoholic  solution  of  the  pigment  of  either 
zone. 

TABLE  9.     ABSORPTION  BANDS  OF  PIGMENTS  OF  COLOSTRUM  MILK  FAT. 


Combined  Pigment 
(CS2  Solution) 

Carotin 
(CS2  Solution) 

Xanthophylls 

(CS2  Sol.) 

(C2HBOH  Sol.) 

Band  I 
Band  II 
Band  III 

223-240 
260-278 
300-320 

224-242 
259-278 
302-319 

232-249 
272-291 
312-330 

264-281 
306-326 
356-.  .  . 

Crystalline  Form  of  Carotin  From  Butter  Fat. — The  great  concen- 
tration of  pigment  in  the  fat  from  colostrum  milk  seemed  to  offer 
an  excellent  opportunity  to  at  least  attempt  the  isolation  of  the  pig- 


360    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 

ment  in  crystalline  form.  The  pigment  was  isolated  in  the  usual 
way  from  forty  grams  of  very  high  colored  colostrum  fat  from  a 
Jersey  cow.  Great  care  was  taken  during  the  isolation  to  avoid 
aldehyde  resin  pigments.  This  was  successful.  The  ether  solution 
of  the  unsaponifiable  substances  was  evaporated  at  35°  C.  and  the 
residue  dissolved  at  once  in  carbon  bisulphide.  This  solution,  which 
had  a  blood  red  color,  was  concentrated  to  2  c.  cm.  volume  at  a  low 
temperature  and  an  excess  of  cold  absolute  alcohol  added.  There 
was  no  immediate  crystallization,  but  after  standing  several  days 
there  were  deposited  a  number  of  yellow  crystals.  These  were  at 
first  thought  to  be  crystals  of  the  pigment,  for  similar  crystals  were 
obtained  in  the  same  manner  from  the  corpus  luteum  pigment.  The 
crystals  were  perfectly  formed  double  pyramidal  forms,  but  proved 
to  be  crystals  of  sulphur  which  evidently  arose  from  the  carbon  bisul- 
phide used.  A  further  attempt  at  crystallization  of  the  pigment  was 
abandoned.  It  undoubtedly  would  prove  successful  if  sufficient  pure 
material  could  be  obtained. 

THE  RELATION  BETWEEN  THE  COLOR  OF  THE  MILK  FAT 
AND  THE  FOOD  OF  THE  COW. 

General  observation  for  no  doubt  hundreds  of  years,  at  least, 
ever  since  butter  has  become  of  importance  in  the  diet  of  man,  has 
shown  that  green  feeds  of  all  kinds,  especially  fresh  green  grass 
greatly  increase  the  color  of  butter  fat.  Other  feeds,  such  as  car- 
rots, beets  and  yellow  corn  have  been  said  to  have  the  same  effect. 
It  has  never  been  the  subject  of  a  scientific  investigation  however, 
to  show  just  what  relation  exists  between  the  food  of  the  cow  and 
the  color  of  the  milk  fat.  With  a  chemical  relation  established  be- 
tween the  milk  fat  pigments  and  carotin  and  xanthophylls,  the  rela- 
tion of  the  color  of  the  milk  fat  to  the  food  seems  to  be  readily 
explained  on  the  ground  that  the  foods  that  have  been  observed  to 
cause  the  highest  colored  butter,  namely,  green  foods,  carrots,  etc. 
are  those  which  are  especially  rich  in  carotin  and  xanthophylls,  par- 
ticularly carotin,  as  in  the  case  of  the  carrots.  Indeed  we  can  go 
still  further  and  definitely  state  that  these  pigments  must  be  abund- 
antly present  in  the  food  before  the  milk  fat  will  show  a  high  color, 
as  will  be  demonstrated  by  the  experiments  which  are  about  to  be 
reported. 

Character  of  the  Yellow  Pigments  of  the  Common  Cattle  Feeds. — 
The  chemical  study  of  the  yellow  pigment  of  milk  fat,  shows  that 
its  principal  constituent  belongs  to  the  hydrocarbon  or  carotin  group 
of  pigments,  although  it  also  contains  xanthophylls  as  a  very  minor 
secondary  constituent.  It  accordingly  became  necessary  to  study  the 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT        361 

nature  of  the  pigments  of  various  common  cattle  foods  before  con- 
ducting any  feeding  experiments  to  prove  that  there  is  a  direct  rela- 
tion between  the  presence  of  carotin  in  the  butter  fat  and  the  presence 
of  carotin  in  the  food.  It  was  at  first  merely  sought  to  show  the 
presence  or  absence  of  unsaponifiable  yellow  pigments  in  various 
foods,  by  extracting  them  with  hot  alcohol,  saponifying  the  extract 
with  potassium  hydroxide  and  extracting  the  pigment  from  the  soap 
with  ether.  From  this  standpoint,  it  was  found  that  among  the 
grains  and  concentrates,  wheat  bran,  linseed  meal,  dried  brewer's  grains 
and  cottonseed  meal  all  showed  the  presence  of  small  amounts  of  un- 
saponifiable yellow  pigments,  while  white  corn  was  found  to  be  the  only 
common  grain  absolutely  free  from  such  pigments.  Yellow  corn  was 
of  course  found  to  be  rich  in  unsaponifiable  yellow  pigments.  Some 
roughages  such  as  corn  silage  and  cottonseed  hulls  were  found  to 
contain  small  amounts  of  unsaponifiable  pigments,  while  wheat  straw 
and  oat  straw  were  practically  free  from  them.  The  hays  were 
found  to  be  the  most  variable  of  all  feeds.  All  green  hays,1  such 
as  alfalfa,  first-class  clover  and  the  very  best  timothy  were  found 
to  contain  considerable  amounts  of  unsaponifiable  yellow  pigments, 
the  amounts  varying  with  the  greenness  of  the  hay.  Bleached  hays, 
whether  timothy,  clover,  or  alfalfa  were  more  or  less  free  from  un- 
saponifiable yellow  pigments. 

Some  feeds  were  investigated  more  particularly  with  a  purpose 
of  showing  the  character  of  the  unsaponifiable  yellow  pigments.  With- 
out reporting  the  experimental  details  in  all  cases,  but  merely  stat- 
ing that  the  methods  of  analysis  were  spectroscopic,  chromotographic, 
and  solubility  methods,  the  following  results  were  obtained. 

Cotton  Seed  Meal  and  Cotton  Seed  Hulls. — It  was  found  that  the 
unsaponifiable  yellow  pigments  of  cottonseed  meal  and  cottonseed 
hulls  were  due  entirely  to  the  oil  they  contain;  and  further  that  this 
oil  which  is  known  to  be  characterized  by  its  yellow  color  contains 
equal  proportions  of  carotin  and  xanthophyll.  The  carotin  is  the 
usual  one  met  in  other  places,  while  the  xanthophyll  is  made  up  of 
at  least  five  different  constituents  according  to  their  adsorption  prop- 
erties. The  chief  xanthophyll  is  not  adsorbed  to  any  extent  by  CaCO3 
from  its  carbon  bisulphide  solution,  and  in  this  solvent  shows  absorption 
bands  of  characteristic  position,  shifted  considerably  toward  the  blue 
from  the  normal  xanthophyll  bands.  In  CS2  the  bands  measured: 
I,  238-260;  II,  285-303;  III,  355—.  The  remainder  of  the  xanthophylls 
were  so  firmly  held  by  the  CaCO3  that  they  could  not  be  readily 

1.  By  green^  hay  is  meant  hay  that  has  been  cured  under  such  conditions 
that  it  still  retains  a  large  part  of  the  green  color  which  characterizes  its  uncut 
condition.  The  green  alfalfa  hay  referred  to  throughout  this  paper  was  west- 
ern cured  alfalfa  which  had  a  remarkably  bright  green  color. 


362    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN    NO.    IO 

washed  out  with  a  stream  of  carbon  bisulphide.  They  were  all 
washed  out  however,  by  a  one  per  cent  alcoholic  petroleum  ether 
solution. 

Bleached  Alfalfa  Hay. —  This  hay  was  quite  free  from  green 
stalks  and  as  it  had  been  found  palatable  to  the  cows  it  was  of  special 
interest  as  an  experimental  roughage  for  non-pigmented  feeding 
studies.  A  selected  sample  of  the  hay  was  ground  up  fine,  and 
extracted  with  ten  per  cent  alcoholic  petroleum  ether.  The  light 
grqen  colored  extract  showed  no  indication  of  either  carotin  or 
xanthophyll  when  analyzed  by  means  of  the  chromotogramm,  and  no 
yellow  pigment  was  extracted  from  its  alcoholic  solution  by  petro- 
leum ether  or  by  carbon  bisulphide. 

Yellow  Corn. — The  unsaponifiable  yellow  pigment  of  yellow  corn 
is  in  all  probability  in  the  oil.  It  was  found  to  be  composed  of  two 
constituents,  the  largest  part  of  which  is  a  xanthophyll-like  pigment, 
showing  absorption  bands  in  alcoholic  and  carbon  bisulphide  solu- 
tions lying  close  to  the  normal  xanthophyll  bands.  In  CS2  the  bands 
measured:  I,  229-251;  II,  274-291;  III,  not  measured.  It  was  not 
adsorbed  from  either  petroleum  ether  or  carbon  bisulphide  by  CaCO3 
but  passed  through  as  a  yellow  or  orange  zone.  Its  carbon  bisulphide 
solutions  were  orange  colored.  Petroleum  ether  readily  extracted 
the  pigment  from  its  concentrated  eighty  per  cent  alcoholic  solu- 
tion, but  it  could  be  completely  re-extracted  from  its  petroleum  ether 
solution  by  fresh  eighty  per  cent  alcohol.  In  this  respect  it  differed 
from  any  xanthophyll-like  pigment  yet  investigated.  The  pigment 
was  more  soluble  in  carbon  bisulphide  than  in  eighty  per  cent  alcohol 
and  in  this  respect  favored  carotin.  On  warming  its  alcoholic  solu- 
tion containing  a  little  concentrated  hydrochloric  acid,  the  color  of 
the  solution  changed  to  a  light  greenish  blue,  with  the  fading  of 
the  absorption  bands.  The  minor  constituent  of  the  corn  pigment 
had  the  spectroscopic,  solubility  and  adsorption  properties  of  carotin. 

The  Carrot. — It  was  planned  to  conduct  some  feeding  experiments 
with  carrots,  and  a  special  study  of  its  relative  proportion  of  carotin  and 
xanthophyll  was  accordingly  considered  advisable.  A  large  well- 
colored  carrot  was  scraped,  chopped  fine,  and  boiled  in  water  for 
one  hour.  The  softened  tissue  was  poured  onto  cheesecloth  and 
the  water  squeezed  out  of  the  pulp.  The  pulp  was  pressed  through 
a  wire  gauze,  dried  and  powdered.  The  meal  was  extracted  with 
carbon  bisulphide  and  a  portion  of  the  blood  red  solution  analyzed 
by  means  of  a  chromotogramm.  By  far  the  greatest  part  of  the  pig- 
ment passed  through  unadsorbed  as  a  rose  colored  zone,  leaving  a 
small  amount  of  adsorbed  pigment  in  the  column  which  was  not 
differentiated  into  zones  but  which  was  readily  washed  out  with  alco- 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK  FAT         363 


holic  petroleum  ether.  The  proportion  of  xanthophyll  to  the  entire  pig- 
ment in  this  test  was  very  small,  not  over  three  or  four  per  cent. 

The  rose  colored  carotin  solution  showed  three  beautiful  ab- 
sorption bands,  all  of  equal  intensity.  The  measurements  of  the 
bands  were  completely  in  accord  with  the  standards  given  in  Table  6. 
The  remainder  of  the  carbon  bisulphide  extract  of  the  dried  car- 
rot meal  was  evaporated  into  alcohol.  The  alcohol  was  diluted  with 
water  to  an  eighty  per  cent  solution  and  extracted  with  petroleum 
ether  (b.  p.  3O°-5o°  C)  until  no  more  color  was  extracted.  The  alco- 
holic solution  of  the  xanthophylls  which  remained  was  combined  with 
the  solution  of  the  xanthophylls  washed  out  of  the  chromotogramm 
with  alcoholic  petroleum  ether.  The  combined  solutions  were  evapor- 
ated to  dryness  and  the  residue  dissolved  in  a  small  amount  of  carbon 

bisulphide.  This  solution  showed  three 
absorption  bands  and  end  absorption.  The 
measurements  were  the  same  as  given  in 
Table  No.  6. 

This  carbon  bisulphide  solution  was 
orange    colored    when    concentrated  and 
gave  the  chromotogramm  shown  in  fig- 
ure IV.  There  was  a  small  amount  of  car- 
otin pigment  present  which  being  unad- 
sorbed  was  readily  washed  out  and  caught 
at  the  lower  end  of  the  tube.    Zone  VIII 
zone  ii:  Orange.  was  aiso  on\y  siightly  adsorbed  and  was 
colorless.  washed  out  by  a  stream  of  carbon  bisul- 

-  zone  ni:  orange,  phide.     When  this  zone  was  washed  out 
the  suction    was    stopped.     The    CaCO3 
tinted  zones  were  then  removed  separately 
from  the  column  with  the  aid  of  a  knife; 
52  was  drawn  off  in  each  case  at  a 


Colorless. 


Zone  I:    Yellow. 


Colorless. 


colorless. 


Colorless. 


Zone  V:  ,*        ^o 

Yellow-Orange,  the   Cb, 


zonevi*  Green    ^ow  temPerature ?  an^  the  pigment  washed 

Colorless. 

/  Zone  VII: 
\  Red-Orange. 

Colorless. 


7 


f  Zone  VIII: 
\  Faint  Red 
(  Orange. 


Rose. 


Cotton  Plug. 


FIGURE  IV. 


out  with  ninety-five  per  cent  alcohol. 

Zone  II  was  found  to  contain  the 
most  pigment,  judged  from  the  color  of 
its  alcoholic  solution,  with  the  lower  zones 
about  the  same  with  the  exception  of  Zone 
V  which  had  very  little  pigment.  Zone 
I  contained  considerably  more  pigment 
than  was  indicated  by  the  color  of  its 
zone  in  the  chromotogramm.  The  spec- 
troscopic  properties  of  all  the  xanthophyll 
constituents  were  studied  with  respect  to 
the  normal  xanthophyll  bands.  The  results 


364   MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 


are  shown  in  Table  10.  The  observations  were  not  made  by  observing 
all  the  pigments  in  the  same  volume  but  the  volumes  were  adjusted 
in  each  case  to  give  the  best  possible  bands. 

It  will  be  noticed  that  with  the  exception  of  Zone  I,  all  the  xan- 
thophylls  showed  the  three  normal  xanthophyll  bands  to  some  degree  of 
intensity.  Similarly  hydrochloric  acid  had  no  effect  on  any  of  these  so- 
lutions or  on  the  bands.  Nitric  acid  had  the  same  effect  on  the  pigments 
of  Zones  II,  VII,  and  VIII  causing  the  solutions  to  fade  with  the  disap- 
pearance of  the  bands.  The  effect  of  nitric  acid  on  the  pigments  of 
Zones  III  and  IV  was  somewhat  different.  In  these  two  cases  a  fourth 
well-developed  band  appeared  before  the  solution  lost  its  color  and 
the  three  normal  bands  faded.  The  pigment  of  Zone  I  was  entirely 
different  from  any  of  the  others.  The  normal  first  band  of  the 
xanthophylls  was  entirely  missing  and  the  alcoholic  solution  turned 
a  distinct  bluish  green  on  the  addition  of  a  little  concentrated  hy- 
drochloric acid.  This  color  persisted  for  24  hours,  long  after  the 
absorption  bands  had  disappeared.  This  was  evidently  the  xanthophyll 
B  of  Tswett  and  the  xanthophyll  B  of  C.  A.  Schunck. 


TABLE  No.  10. 


SPECTROSCOPIC  PROPERTIES  OF  THE  XANTHOPHYLLS  OF  THE 
CARROT. 


Zone 

Band  I 

Band  II 

Band   III 

End  Absorption 

I 
II 

Missing 
Fair 

Good 
Fair 

Good 
Fair 

Very  faint 
None 

III 

Very  strong 

Very  strong 

Weak 

None 

IV 
V 

Strong 
Very  faint 

Very  strong 
Good 

Faint 
Good 

None 
None 

VII 
VIII 

Very  strong 

Very  strong 
Good 

Very  faint 
Very  faint 

None 
Very  faint 

THE  FEEDING  EXPERIMENTS. 

The  foregoing  studies  indicated  that  the  foods  best  adapted 
for  non-pigmented  rations  were  bleached  hays  and  cottonseed  hulls 
for  roughages  and  white  corn  and  cottonseed  meal  for  grains.  To 
study  the  variation  in  the  color  of  butter  fat,  the  ration  of  various 
cows  was  changed  to  one  containing  the  smallest  possible  amounts 
of  carotin  and  xanthophylls,  and  the  butter  fat  studied  colorimetrically 
during  this  time.  The  procedure  for  the  butter  fat  was  in  each  case 
as  follows :  The  milk  of  one  or  two  milkings  was  separated  by  means 
of  a  centrifugal  hand  separator  and  the  cream  churned  by  hand  in 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK  FAT         365 

quart  bottles.  The  butter  thus  obtained  was  rendered  at  a  tempera- 
ture of  50°  to  60°  C.  and  the  rendered  fat  filtered.  The  pure  filtered 
fat  was  analyzed  colorimetrically  by  means  of  the  Lovibond  tintometer 
and  its  standard  color  glasses.  The  color  of  the  fat  was  always  com- 
pared in  one-inch  layer.  The  Lovibond  tintometer  is  shown  in  Fig- 
ure V. 


FIGURE  V. 

The  solution  (in  the  present  case  melted  butter  fat)  whose  color 
is  to  be  measured  is  placed  in  a  cell  with  glass  ends  (one  inch  apart 
in  all  this  work),  and  the  color  matched  by  standard  color  glasses 
of  various  units  of  yellow,  red  or  blue,  and  the  color  of  the  solution 
read  by  adding  together  the  various  glasses  of  color  used  to  match 
the  unknown  color.  Melted  butter  fat  having  an  orange  tint  requires 
only  yellow  and  red  to  match  its  color.  All  readings  are  made  with 
the  instrument  pointing  towards  the  daylight  (not  sunlight).  The 
instrument  is  quite  sensitive  towards  the  yellow  glasses  below  25  units 
of  yellow  but  the  sensitiveness  decreases  considerably  above  40  units 
of  yellow.  In  other  words,  it  is  possible  to  match  the  exact  color  of 
an  "unknown"  much  more  closely  when  its  color  is  below  30  to  35 
units  of  yellow  than  when  its  color  is  above  this  value.  In  a  great 
many  cases,  and  this  nearly  always  applies  to  butter  fat,  it  is  possible 
to  match  the  tint  of  the  fat  but  the  color  of  the  fat  is  more  brilliant 
than  that  of  the  combined  standard  glasses.  In  this  case  an  exact 
match  can  be  obtained  by  "damping  down"  the  butter  fat  color  by 
inserting  in  front  of  it  equal  units  of  the  three  colors,  yellow,  red 
and  blue,  and  recording  this  as  "light." 

Before  reporting  the  data  dealing  with  the  variation  in  the  color 
of  the  butter  fat  it  may  be  possible  to  convey  some  idea  of  what  the 
various  colors  mean  when  applied  to  butter  fat  by  stating  that  ren- 
dered "June"  butter  in  the  one-inch  cell  will  give  a  color  of  from  80 
to  60  units  of  yellow.  Color  readings  between  45  and  25  units  of 


366   MISSOURI  AGRICULTURAL  EXP.   STAV   RESEARCH   BULLETIN   NO.    IO 

yellow  would  accordingly  indicate  a  fairly  well  colored  to  light  col- 
ored butter,  between  20  and  8  units  of  yellow  would  be  called  light 
to  very  light  colored  butter,  while  below  these  limits  ranging  down 
to  i  or  2  units  of  yellow  would  be  called  white  to  "dead"  white, 
especially  if  the  fat  was  still  in  the  form  of  butter. 

Experiment  i. 

The  ration  of  Cow  No.  57,  a  pure  bred  Jersey,  was  changed 
from  a  ration  rich  in  carotin  and  xanthophylls  to  a  ration  containing 
a  very  small  amount  of  these  pigments.  The  ration  rich  in  carotin 
and  xanthophylls  consisted  of  alfalfa  hay  and  yellow  corn.  The  ration 
poor  in  these  pigments  was  composed  of  bleached  clover  hay  and 
white  corn.  The  results  of  the  experiment  are  shown  in  Table  n. 

The  change  from  a  ration  rich  in  carotin  and  xanthophylls  to 
one  poor  in  these  pigments  caused  the  color  of  the  butter  fat  to  drop 
from  43  units  of  yellow  to  8.5  units  of  yellow,  from  a  well  colored 
to  a  very  light  colored  fat.  This  change  of  color  was  very  gradual 
and  required  29  days.  It  should  be  stated,  however,  that  the  cow 
did  not  relish  her  non-pigmented  ration.  She  lost  weight  regularly, 
and  her  milk  production  fell  off  a  great  deal.  It  was  apparent  that 
the  animal  was  drawing  heavily  during  this  entire  period  from  a 
storage  of  pigment  in  her  body.  It  will  be  shown  in  the  subsequent 
papers  of  this  series  that  in  this  experiment  the  blood  and  also  the 
body  fat  were  supplying  the  pigments  for  the  milk  fat. 

It  may  be  stated  that  a  slow  lowering  of  the  color  of  the  milk 
fat,  such  as  took  place  in  this  experiment,  would  be  normal  for  all 
Jersey  cows  whose  ration  is  changed  to  an  unpalatable,  non-pigmented 
one  like  that  used  in  this  experiment.  The  explanation  for  this  is 
found  in  the  high  color  of  the  body  fat  of  this  breed  of  cows.  We 
therefore  have  here  a  clear  explanation  of  why  Jersey  cows  will  some- 
times apparently  give  yellow  milk  fat  during  the  winter  months  when 
their  food  is  almost  or  entirely  lacking  in  carotin  and  xanthophylls. 
Under  these  conditions  if  the  body  fat  is  called  upon  to  supplement 
the  digestion  products  of  the  food  in  the  production  of  milk  fat  at 
the  same  time  the  blood  serum  storage  of  pigments  is  being  drawn 
upon,  it  is  clear  that  the  reduction  in  color  of  the  milk  fat  will  be  very 
gradual,  as  in  the  case  of  Cow  No.  57,  and  a  complete  elimination  of 
color  may  require  a  long  period  of  time. 

Continuing  the  discussion  of  the  experiment  it  is  seen  that  when 
the  color  of  the  milk  fat  had  dropped  to  8.5  units  of  yellow  the  white 
corn  in  the  ration  was  replaced  by  mixed  corn,  white  and  yellow 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK  FAT         367 

(mostly  yellow).  Later  this  was  replaced  by  yellow  corn  entirely. 
The  roughage  remained  the  same,  i.  e.  bleached  clover  hay.  The 
table  shows  that  yellow  corn  had  no  effect  whatever  upon  the  color 
of  the  milk  fat.  There  seemed  to  be  a  very  slight  effect  at  first  but 
the  color  of  the  fat  soon  dropped  back  to  8.0  units  of  yellow. 


TABLE  No.  10.     THE  EFFECT  OF  A  CAROTIN  AND  XANTHOPHYLL-FREE  RATION 
ON  THE  COLOR  OF  MILK  FAT.     JERSEY  Cow  No.  57. 


Pounds 

Color  of  butter  fat. 

Date 

hay 

Pounds  grain  fed 

per 
day 

per  day 

Yellow 

Red 

Light 

1912 

Corn 

Mixture 

Mar.    1 

15  (a) 

8 

43.0 

2.0 

0.2 

2 

15(b) 

8 

3 

15 

4  white 

4 

4 

15 

4  white 

4 

5 

15 

4  white 

4 

6 

15 

8  white 

7 

15 

8  white 

33.0 

2.0 

0.2 

8 

15 

8  white 

9 

15 

8  white 

29.0 

1.7 

0.2 

10 

15 

8  white 

11 

15 

8  white 

33.0 

1.8 

0.2 

12 

15 

8  white 

26.0 

1.6 

0.2 

13 

15 

8  white 

26.0 

1.7 

0.2 

14 

15 

8  white 

22.0 

2.0 

0.2 

15 

15 

8  white 

22.0 

2.0 

0.2 

16 

15 

8  white 

21.0 

1.8 

0.2 

17 

15 

8  white 

18 

15 

8  white 

18.0 

1.7 

0.2 

19 

15 

8  white 

19.0 

1.6 

0.2 

20 

15 

8  white 

18.0 

1.6 

0.2 

21 

15 

8  white 

22 

15 

8  white 

12.0 

1.6 

0.2 

23 

15 

8  white 

24 

7 

10  white 

25 

7 

10  white 

11.0 

1.6 

0.2 

26 

7 

10  white 

11.0 

1.6 

0.2 

27 

7 

10  white 

10.0 

1.5 

0.1 

28 

7 

10  white 

10.0 

1.5 

0.1 

29 

7 

10  white 

8.5 

1.8 

0.2 

30 

7 

10  white 

9.0 

1.7 

0.2 

31 

7 

10  mixed 

10.0 

1.8 

0.2 

(a)     Alfalfa. 

(b)     Clover—  Mar.  2  to  Mar.  31. 

The  7  Ibs.  of  clover  hay  was  now  replaced  by  10  Ibs.  of  alfalfa 
hay  rich  in  carotin  and  xanthophylls.    The  effect  on  the  color  of  the 


368   MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 


TABLE  11  (CONTINUED).     EFFECT  OF  FEEDING  YELLOW  CORN  TO  A  Cow  GIV- 
ING Low  COLORED  MILK  FAT.     JERSEY  Cow  No.  57. 


Date 

Pounds 
hay 
per 
day 

Pounds 
grain   fed 

day 

Color  of  butter  fat 

Yellow 

Red 

Light 

1912 

Corn 

Apr.    1 

7  (a) 

10  mixed 

8.5 

1.7 

0.2 

2 

7 

10  mixed 

8.0 

2.1 

0.2 

3 

7 

10  mixed 

8.0 

.9 

0.2 

4 

7 

10  mixed 

12.0 

.5 

0.2 

5 

7 

10  mixed 

12.0 

.7 

0.2 

6 

7 

10  mixed 

11.0 

.8 

0.2 

7 

7 

10  yellow 

8 

7 

10  yellow 

11.0 

.8 

0.2 

9 

7 

10  yellow 

8.0 

.7 

0.2 

10 

7 

10  yellow 

9.0 

.7 

0.2 

11 

10(b) 

10  yellow 

10.0 

.5 

0.2 

12 

10 

10  yellow 

12.0 

.6 

0.2 

13 

10 

10  yellow 

15.0 

.6 

0.2 

14 

10 

10  yellow 

20.0 

.6 

0.2 

15 

10 

10  yellow 

33.0 

.7 

0.2 

16 

10 

10  yellow 

36.0 

.6 

0.2 

17 

10 

10  yellow 

38.0 

.6 

0.2 

18 

10 

10  yellow 

43.0 

.6 

0.2 

19 

10 

10  yellow 

45.0 

2.0 

0.5 

21 

10 

10  yellow 

43.0 

1.8 

0.5 

22 

10 

10  yellow 

43.0 

1.7 

0.5 

23 

10(c) 

10  yellow 

46.0 

1.7 

0.5 

24 

10 

10  yellow 

40.0 

1.8 

0.5 

25 

10 

10  yellow 

43.0 

1.8 

0.5 

27 

10 

10  yellow 

52.0 

2.0 

0.5 

30 

10 

10  yellow 

57.0 

2.1 

0.5 

May   2 

10 

10  yellow 

64.0 

2.1 

0.5 

4 

10 

10  yellow 

64.0 

2.2 

0.5 

5-10(d) 

Pasture  only 

80.0 

2.5 

0.5 

a)  Clover— Apr.  1-10. 

b)  Alfalfa— Apr.  11-22. 

(c)  Alfalfa  and  pasture — Apr.  23-May  10. 

(d)  Sample  taken  May  10. 

milk  fat  was  immediate.  At  the  end  of  seven  days  the  color  had 
increased  to  43  units  of  yellow,  the  maximum  supplied  by  this  rough- 
age. Later  the  ration  was  supplemented  by  some  fresh  pasture  grass. 
The  color  of  the  milk  fat  increased  to  64  units  of  yellow.  Still  later 
the  cow  was  turned  out  to  pasture  alone.  The  color  then  reached 
the  maximum  we  have  observed,  i.  e.  80  units  of  yellow. 

Experiment  No.  2. 

This  experiment  was  conducted  with  Cow  No.  301,  a  pure  bred 
Ayrshire.    A  short  time  previous  to  the  experiment  here  reported  this 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF   MILK  FAT         369 

cow  was  in  very  poor  flesh  resulting  from  a  feeding  experiment  in 
which  she  was  heavily  underfed.  Her  ration  during  her  underfeeding 
was  composed  of  alfalfa  hay  rich  in  carotin  and  xanthophylls  and  a 
grain  mixture  of  corn,  bran  and  linseed  meal.  At  the  end  of  the 
underfeeding  experiment  the  ration  was  changed  to  bleached  alfalfa 
hay,  the  pigmentation  of  which  was  reported  above,  and  white  corn 
and  cottonseed  meal.  Enough  of  this  ration  was  given  to  bring  the 
cow  back  to  normal  feeding  conditions.  At  the  end  of  this  time  the 
experiment  here  reported  was  begun.  The  results  are  given  in 
Table  12. 

At  the  end  of  ten  days  the  color  of  the  milk  fat  had  dropped  to 
9  units  of  yellow.  When  it  was  apparent  after  a  week's  further  trial 
that  the  color  had  reached  at  least  a  temporary  minimum  value  for 
this  ration,  the  grain  was  changed  to  yellow  corn  entirely.  This  was 
finally  increased  to  12  Ibs.  per  day.  The  result  was  very  clearly  in 
accord  with  that  of  Experiment  No.  I,  showing  that  yellow  corn  is 
not  a  source  of  pigment  for  the  milk  fat  of  dairy  cows. 

TABLE  No.  12.     EFFECT  OF  NON-PIGMENTED  RATION  AND  A  RATION  CONTAIN- 
ING YELLOW  CORN  UPON  THE  COLOR  OF  MILK  FAT.    AYR- 
SHIRE Cow  No.  301. 


Date  of 

Pounds 
alfalfa 

Pounds 

Pounds 
cotton- 

Color of  1 

cutter  fat 

sample 

hay 

corn 

seed 
meal 

Yellow 

Red 

1912 

October        3 

16 

6(a) 

2 

27.0 

.7 

13 

16 

6 

2 

9.0 

.7 

17 

16 

6 

2 

6.0 

.5 

21 

16 

6 

2 

9.0 

.2 

24 

16 

8(b) 

7.5 

.2 

25 

16 

8 

9.0 

.5 

26 

16 

8 

10.0 

1.5 

27 

16 

8 

9.0 

1.5 

28 

16 

8 

9.0 

1.5 

29 

16 

8 

10.0 

1.5 

30 

16 

8 

8.5 

1.2 

31 

16 

8 

10.0 

1.2 

November    1 

16 

12 

9.5 

1.2 

2 

16 

12 

10.0 

1.2 

3 

16 

12 

8.5 

1.2 

4 

16 

12 

8.0 

1.2 

(a)  Oct.  3-21,  white  corn. 

(b)  Oct.  24- Nov.  4,  yellow  corn. 


370    MISSOURI   AGRICULTURAL  EXP.   STAV   RESEARCH   BULLETIN   NO.    IO 

Experiment  No.  3. 

This  feeding  experiment  was  conducted  with  the  same  cow  as 
the  preceding  experiment  and  immediately  followed  that  experiment. 

It  seemed  very  probable  that  the  reason  the  color  of  the  milk 
fat  in  Experiments  i  and  2  could  not  be  lowered  more  than  the  uni- 
form figure  found  in  both  experiments,  i.  e.,  about  8  units  of  yellow, 
was  due  to  the  fact  that  the  ration  was  supplying  a  small  amount  of 
pigment  and  also  to  the  fact  that  the  normal  storage,  that  in  the 
blood  serum,  had  not  been  exhausted.  It  seemed  reasonable  to  sup- 
pose, therefore,  that  if  the  first  factor  was  eliminated  at  the  outset, 
the  second  would  necessarily  also  be  eliminated  if  the  experiment  was 
continued  for  a  sufficient  length  of  time.  The  experiment  here  re- 
ported was  for  the  purpose  of  testing  the  validity  of  this  supposition, 
and  also  for  the  purpose  of  ascertaining  to  how  low  a  point  the  color 
of  the  milk  fat  could  be  reduced.  The  ration  chosen  was  one  which 
would  supply  the  least  amount  of  carotin  and  xanthophylls.  It  was 
composed  of  cottonseed  meal  and  cottonseed  hulls.  The  results  of 
the  experiment  are  given  in  Table  13. 

The  supposition  stated  above  was  fully  borne  out  by  the  long 
continued  feeding  of  a  practically  non-pigmented  ration.  At  the  end 
of  52  days'  feeding,  the  cow  was  producing  absolutely  colorless  butter. 
It  was  only  when  the  rendered  butter  was  viewed  in  the  tintometer 
that  a  very  slight  amount  of  color  could  be  detected.  This  very 
slight  amount  of  color  was  due  to  the  fact  that  the  normal  storage 
of  pigment  in  the  body,  that  in  the  blood  serum,  had  not  been  com- 
pletely exhausted,  as  will  be  shown  in  a  subsequent  paper  of  this 
series.  It  is  probable  also  that  the  body  was  being  drawn  upon  for 
some  of  its  pigments,  for  the  animal  suffered  somewhat  from  under- 
feeding on  this  ration.  It  is  also  possible  that  a  very  small  amount 
of  carotin  was  being  supplied  by  the  oil  in  the  cottonseed  meal  and 
hulls.  For  practical  purposes  such  a  supply  of  pigment  would  of 
course  be  considered  absolutely  negative. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF  MILK  FAT         37! 


TABLE  No.  13.     EFFECT  OF  A  LONG-CONTINUED  FEEDING  OF  A  NON-PIGMENTED 

RATION  UPON  THE  COLOR  OF  MILK  FAT.     AYRSHIRE 

Cow  No.  301. 


Date  of 
sample 

Pounds 
alfalfa 
hay 

Pounds 
cotton- 
seed 
hulls 

Pounds 
cotton- 
seed 
meal 

Pounds 
Corn 

Color  of  butter  fat 

Yellow 

Red 

1912-13 

Nov.        5 

6.5 

12 

9.0 

1.2 

6 

4.5 

0.5 

0.5 

10 

8.0 

1.2 

7 

8.0 

1.0 

1.0 

8 

9.0 

1.1 

8 

8.0 

2.0 

2.0 

7 

8.0 

1.0 

9 

8.0 

2.0 

2.0 

6 

7.0 

1.0 

10 

7.0 

3.0 

3.0 

6 

6.5 

1.0 

11 

6.0 

3.0 

3.0 

5 

6.0 

1.0 

12 

6.0 

4.0 

4.0 

4 

5.5 

0.9 

13 

6.0 

4.0 

4.0 

4 

5.0 

0.8 

14 

3.0 

5.0 

5.0 

2 

5.0 

0.8 

15 

2.0 

6.0 

6.0 

5.0 

0.9 

16 

10.0 

6.0 

4.0 

0.7 

17 

12.0 

6.0 

4.5 

0.8 

18 

12.0 

7.0 

4.0 

0.7 

19 

12.0 

7.0 

3.5 

0.7 

20 

12.0 

8.0 

3.5 

0.7 

21 

13.0 

8.0 

3.5 

0.7 

22 

14.0 

8.0 

3.5 

0.7 

23 

14.0 

8.0 

3.2 

0.7 

24 

16.0 

8.0 

3.1 

0.7 

25 

16.0 

8.0 

3.0 

0.7 

26 

16.0 

8.0 

2.6 

0.5 

27 

16.0 

8.0 

3.2 

0.7 

28 

16.0 

8.0 

2.5 

0.7 

29 

*  , 

16.0 

8.0 

3.0 

0.7 

Jan.          2 

16.0 

8.0 

1.4 

0.5 

7 

16.0 

8.0 

1.3 

0.4 

It  will  be  of  interest  to  state  in  connection  with  this  experiment 
that  it  furnished  the  three  "light  colored"  fats  for  the  studies  of  the 
proportion  of  carotin  and  xanthophyll  which  were  reported  above. 

Experiment  4. 

The  color  of  the  butter  'fat  of  Cow  No.  301  was  now  so  low  that 
the  conditions  were  considered  ideal  for  one  or  two  additional  im- 
portant investigations,  first  a  confirmation  of  the  apparently  negative 
effect  of  feeding  yellow  corn  which  was  obtained  in  Experiments  I 
and  2,  and  second  a  study  of  carrot  feeding.  In  the  latter  study  the 
results  would  be  especially  interesting  in  view  of  the  fact  that  the 
pigment  fed  would  be  almost  pure  carotin.  The  hay  used  in  this 
experiment  was  a  very  light  colored  timothy  hay  which  was  not  quite 
so  free  from  carotin  and  xanthophylls  as  the  bleached  alfalfa  of  Expe- 
riment 3,  but  which  apparently  had  no  effect  on  the  color  of  the  fat 


372    MISSOURI   AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 


as  the  data  will  show.     The  results  of  the  two  studies  are  given  in 
Table  14. 

In  regard  to  yellow  corn  feeding,  it  is  seen  that  replacing  2  Ibs. 
cottonseed  meal  and  4  Ibs.  white  corn  by  6  Ibs.  yellow  corn  had  no 
influence  upon  the  color  of  the  milk  fat.  If  yellow  corn  has  any  value 
as  an  aid  in  the  production  of  yellow  milk  fat,  it  would  certainly 
have  been  evident  in  this  experiment  where  the  milk  fat  was  prac- 
tically colorless  before  feeding  the  yellow  corn.  This  experiment 
therefore  gives  conclusive  proof  of  the  negative  value  of  yellow  corn 
in  the  pigmentation  of  milk  fat.  The  reason  for  this  will  be  discussed 
more  fully  in  the  general  discussion  of  the  experiments. 

TABLE  No.  14.     EFFECT  OF  A  RATION  CONTAINING  YELLOW  CORN  AND  OF  A 
RATION  CONTAINING  CARROTS  UPON  THE  COLOR  OF  MILK  FAT. 
AYRSHIRE  Cow  No.  301. 


Date  of 

Pounds 
rough- 

Pounds 
carrots 

Pounds 
cotton- 

Pounds 
corn 

Color  of  1 

Dutter  fat 

sample 

age(a) 

seed  meal 

Yellow 

Red 

1912 

Jan.         3 

14 

4 

4  white 

21 

12 

4 

4  white 

24 

12 

4 

4  white 

1.2 

0.4 

25 

12 

4 

4  white 

28 

12 

2 

6  yellow 

29 

12 

2 

6  yellow 

1.4 

0.5 

30 

12 

2 

6  yellow 

1.5 

0.4 

31 

12 

2 

6  yellow 

1.7 

0.4 

Feb.         1 

12 

2 

6  yellow 

1.7 

0.4 

2 

12 

2 

6  yellow 

2.0 

0.5 

3 

12 

2 

6  yellow 

1.8 

0.5 

4 

12 

2 

6  yellow 

2.0 

0.5 

5 

12 

2 

6  yellow 

1.8 

0.3 

6 

12 

2 

6  yellow 

2.0 

0.5 

7 

12 

6 

4 

4  white 

2.2 

0.5 

8 

12 

20 

4 

4  white 

2.2 

0.5 

9 

12 

30 

4 

4  white 

2.3 

0.5 

10 

12 

40 

4 

4  white 

3.2 

0.6 

11 

12 

40 

4 

4  white 

4.5 

0.7 

12 

12 

50 

4 

4  white 

5.5 

1.0 

13 

12 

50 

4 

4  white 

8.0 

1.0 

14 

12 

50 

4 

4  white 

11.5 

.0 

15 

12 

30 

4 

4  white 

16.0 

.1 

16 

12 

30 

4 

4  white 

15.0 

.2 

17 

12 

20 

4 

4  white 

18.0 

.3 

18 

20 

19.0 

.  1 

19 

3 

15.0 

.1 

20 

5 

10.0 

.3 

21 

3 

10 

1 

1  white 

8.0 

.2 

22 

3 

10 

2 

2  white 

9.0 

.3 

(a)     Roughage  consisted  of  2  parts  bleached  timothy  hay  and  1  part  cotton- 
seed hulls. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF  MILK  FAT         3/3 
TABLE  No.  14.     (CONTINUED) 


Date  of 

Pounds 
rough- 

Pounds 

Pounds 
cotton- 

Pounds 

Color  of  1 

cutter  fat 

sample 

age(a) 

carrots 

seed 
meal 

corn 

Yellow 

Red 

1913 

Feb.       23 

12 

20 

4 

4  white 

11.0 

.3 

24 

12 

20 

4 

4  white 

21.0 

.3 

25 

12 

20 

4 

4  white 

28.8 

.2 

26 

12 

20 

4 

4  white 

36.0 

.8 

27 

12 

20 

4 

4  white 

27.0 

.3 

28 

12 

20 

4 

4  white 

24.0 

.3 

Mar.        1 

12 

20 

4 

4  white 

24.0 

.3 

2 

12 

20 

4 

4  white 

28.0 

.4 

3 

12 

20 

4 

4  white 

26.0 

.4 

4 

12 

20 

4 

4  white 

26.0 

.3 

5 

12 

20 

4 

4  white 

24.0 

.4 

6 

12 

50 

4 

4  white 

24.0 

.3 

7 

12 

4 

4  white 

23.0 

.3 

8 

12 

4 

4  white 

19.0 

.2 

9 

12 

4 

4  white 

19.0 

.2 

10 

12 

4 

4  white 

18.0 

.6 

11 

12 

4 

4  white 

17.0 

.1 

12 

12 

4 

4  white 

14.0 

.1 

13 

14 

4 

4  white 

11.0 

.1 

14 

14 

4 

4  white 

12.0 

.2 

15 

14 

4 

4  white 

12.0 

1.2 

17 

14 

4 

4  white 

10.5 

1.2 

21 

14 

4 

4  white 

7.5 

1.2 

24 

12 

4 

4  white 

7.0 

1.0 

30 

12 

4 

4  white 

7.5 

1.3 

(a)     Roughage  consisted  of  2  parts  timothy  hay  and  1  part  cottonseed  hulls. 

Note:  During  the  period  from  2:18  p.  m.  to  2:23  a.  m.  the  cow  was  badly 
off  feed  and  her  entire  ration  was  withdrawn  for  a  few  days.  She  soon  re- 
covered, however,  and  was  put  back  on  the  experimental  ration,  not  feeding  so 
many  carrots,  however. 

In  regard  to  the  effect  of  feeding  carrots,  the  data  brings  out 
several  interesting  points.  The  carrots  were  added  to  the  ration  on 
February  7,  and  the  amount  was  rapidly  increased  to  50  Ibs.  per  day. 
The  cow  ate  them  with  a  good  deal  of  relish  for  about  a  week  when 
she  began  to  refuse  part  of  them  and  finally  went  off  feed  entirely, 
making  it  necessary  to  withdraw  her  entire  ration  for  a  day  or  two. 
The  effect  of  this  carrot  feeding  period  upon  the  color  of  the  milk 
fat  was  to  increase  the  color  from  1.8  to  19  units  of  yellow.  This  in- 
crease was  not  nearly  as  great,  however,  as  was  expected  considering 
the  large  amount  of  carrots  that  was  fed  and  the  length  of  time  they 
had  been  in  the  ration,  i.  e.  eleven  days. 


374   MISSOURI  AGRICULTURAL  EXP.   STAV   RESEARCH   BULLETIN   NO.    IO 

It  is  clear  from  a  study  of  the  subsequent  data  that  this  abnormal 
result  was  due  to  some  physiological  disturbance  that  finally  resulted 
in  the  cow  going  entirely  off  feed.  As  soon  as  the  carrots  were  re- 
moved from  the  ration,  the  color  of  the  fat  dropped  at  once  to  8  units 
of  yellow.  However,  when  the  animal  had  recovered  from  her  attack 
of  indigestion,  and  the  carrots  were  again  added  to  the  ration,  the 
effect  upon  the  color  of  the  milk  fat  was  perfectly  normal.  On  the 
fourth  day  of  feeding  20  Ibs..  of  carrots  the  color  had  reached  a  max- 
imum of  36  units  of  yellow.  The  color  later  dropped  a  little  with 
an  increase  in  fat  production  which  is  not  shown  in  the  table,  but 
remained  in  the  neighborhood  of  28  units  of  yellow  until  the  carrots 
were  removed.  The  color  then  began  to  drop  slowly  in  the  normal 
way.  After  dropping  to  7  units  of  yellow  on  the  twenty-third  day 
after  the  carrots  had  been  removed,  the  experiment  was  stopped. 

This  experiment  furnished  the  samples  of  butter  fat  whose  pro- 
portion of  carotin  and  xanthophyll  were  studied  and  reported  in  an 
earlier  part  of  this  paper,  namely  the  fat  after  carrot  feeding. 

Experiment  5. 

This  was  a  second  carrot  feeding  experiment  using  another  cow, 
i.  e.  Cow  No.  221,  a  pure  bred  Holstein  cow.  The  experiment  was 
not  as  successful  as  was  hoped  because  of  the  peculiar  appetite  of  the 
cow.  She  refused  to  eat  more  than  ten  pounds  of  the  carrots  per  day 
except  on  two  days  so  the  experiment  was  discontinued.  The  data 
are  given  in  Table  15.  Notwithstanding  the  peculiar  appetite  of  the 
cow  it  is  interesting  to  note  that  the  feeding  of  only  10  Ibs.  of  carrots 
per  day  for  8  days  was  sufficient  to  bring  the  color  of  the  milk  fat 
almost  back  to  the  starting  point,  i.  e.  26  units  of  yellow. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF  MILK  FAT         375 


TABLE  No.  15.     EFFECT  OF  A  NON-PIGMENTED  RATION  AND  OF  A  RATION  CON- 

TAINING  CARROTS  UPON  THE  COLOR  OF  MILK  FAT. 

HOLSTEIN  Cow  No.  221. 


Pounds 

Color  of  butter  fat 

Date  of 

Pounds 

Pounds 

cotton- 

Pounds 

sample 

hay(a) 

carrots 

seed 
meal 

corn 

Yellow 

Red 

1912 
Nov.      29 

Normal  herd  ration  containing  green 
alfalfa  hay. 

26.0 

1.5 

Dec.         9 

16 

4 

4 

8.0 

1.4 

12 

16 

4 

4 

7.0 

1.3 

14 

16 

4 

4 

6.0 

1.1 

17 

16 

10 

4 

4 

6.0 

1.3 

18 

16 

10 

4 

4 

7.0 

.5 

19 

16 

10 

4 

4 

9.0 

.5 

20 

16 

10 

4 

4 

8.0 

.5 

21 

16 

10 

4 

4 

8.0 

.5 

22 

16 

10 

4 

4 

10.0 

.5 

23 

16 

10 

4 

4 

17.0 

.3 

24 

16 

30 

4 

4 

17.0 

.2 

(a.  m.)  25 

16 

20 

4 

4 

14.0 

.5 

(p.  m.)  25 

16 

0 

4 

4 

20.0 

.5 

(a.  m.)  26 

16 

0 

4 

4 

18.0 

.5 

(p.  m.)  26 

16 

0 

4 

4 

17.0 

.5 

27 

16 

0 

4 

4 

18.0 

.5 

28 

16 

0 

4 

4 

12.0 

.5 

1913 

Jan.         3 

16 

0 

4 

4 

7.5 

.5 

22 

Herd  ration  since  1-4-13 

14.0 

.2 

(a)     The  hay  was  a  mixture  of  equal  parts  of  light  colored  timothy  and 
bleached  alfalfa. 

Experiment  No.  6 

This  was  a  feeding  experiment  the  results  of  which  were  ex- 
pected to  corroborate  those  of  Experiment  i,  and  show  that  the  colon 
of  the  milk  fat  of  Jersey  cows  is  as  much  dependent  on  the  food  as 
those  of  other  breeds.  The  variation  in  the  feed  and  the  resulting 
color  of  the  milk  fat  is  shown  in  Table  16.  A  pure  bred  Jersey  cow, 
No.  59,  was  used  for  this  experiment. 


376    MISSOURI   AGRICULTURAL   EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 


TABLE  No.  16.     EFFECT  OF  A  NON-PIGMENTED  RATION  UPON  THE  COLOR  OF 
MILK  FAT.     JERSEY  Cow  No.  59. 


Date  of 
feeding 

Pounds 
grain 
mix- 
ture(a) 

Pounds 
corn 
silage 

Pounds 
hay 

Date     of 
sample 

Color  of  butter  fat 

Yellow 

Red 

Light 

1911 

11.6 
11. 
11. 
11. 
11 
11.5 
12. 

0 
10 
10 
10 

10 

Corn 
Stover 
5 

5 

14(b) 
9(c) 
8 
4 
4 
8(d) 
8 

April      2 
April    14 
April    23 
May      9 
May     20 
June     14 
June    29 

46.0 
6.0 
4.0 
3.0 
3.0 
47.0 
26.0 

1.8 
1.5 
1.4 
1.5 
1.5 
1.8 
2.0 

0.5 
0.2 
0.2 
0.2 
0.2 
0.5 
0.2 

March       11 
to 
April           2 

April           3 
to 
April           4 

April         15 
to 
April         23 

April         24 
to 
May            8 

May          10 
to 
May          20 

May          21 
to 
June          14 

June          15 
to 
June          29 

(a)  The  grain  mixture  was  5  Ibs.  corn  and  6  Ibs.  cottonseed  meal. 

(b)  Green  alfalfa  hay  from  March  11  to  April  2. 

(c)  Bleached  timothy  hay  from  April  3  to  May  20. 

(d)  Green  alfalfa  hay  beginning  May  21. 

The  results  of  this  experiment  show  conclusively  that  Jersey 
cows  are  as  much  dependent  upon  their  food  for  the  pigments  of  the 
milk  fat  as  other  breeds  of  cows.  In  addition  the  experiment  offers 
excellent  proof  of  some  statements  previously  made  in  explanation 
of  the  gradual  lowering  of  the  color  of  the  milk  fat  of  Cow  No.  57 
in  Experiment  No.  i.  It  was  stated  there  that  the  yellow  body  fat 
was  supplementing  the  normal  storage  of  pigment  on  account  of  the 
unpalatableness  of  the  ration.  In  the  present  experiment  we  have  an 
example  of  the  effect  of  changing  the  ration  to  a  non-pigmented  one 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT         377 

without  causing  the  animal  to  draw  upon  any  storage  of  pigment 
other  than  the  normal  one  of  the  blood  serum.  The  result  was  that 
the  color  of  the  milk  fat  dropped  from  46  units  of  yellow  to  6  units 
of  yellow  in  twelve  days,  whereas  in  Experiment  I  it  required  thirty 
days  to  bring  about  a  similar  change  of  color.  The  very  low  color 
of  the  fat  which  was  reached  in  Experiment  6  also  indicates  that 
only  the  normal  storage  was  being  drawn  upon.  Experiment  6  also 
shows  that  corn  silage  is  not  a  source  of  pigment  for  the  milk  fat. 
The  chemical  changes  which  take  place  in  this  roughage  evidently  also 
largely  destroy  the  carotin  and  xanthophylls.  The  chemical  studies  of 
the  pigments  of  corn  silage,  which  were  reported  above,  showed  this  to 
be  the  case. 

RELATION  BETWEEN   COLOR  OF  MILK  FAT  AND  BREED  OF  COW. 

The  foregoing  experiments  have  shown  conclusively  that  dairy 
cows,  exclusive  of  breed,  are  dependent  on  the  carotin  and  xanthophylls 
in  their  feed  for  the  pigment  of  their  milk  fat,  in  other  words,  that 
they  cannot  produce  the  pigment  which  is  thus  secreted.  The  ques- 
tion is  at  once  raised  as  to  wherein  lies  the  so-called  breed  character- 
istic which  is  so  much  emphasized  by  the  breeders  of  Guernsey  and 
Jersey  cattle?  It  will  not  be  denied  that  a  breed  characteristic  does 
exist  in  connection  with  the  color  of  butter  fat.  We  believe,  how- 
ever, that  the  data  now  to  be  presented  will  show  that  this  breed 
characteristic  has  been  overemphasized. 

Since  the  butter  fat  is  dependent  upon  the  food  of  the  cow  for 
its  color,  it  was  necessary  to  compare  the  color  of  the  butter  fat 
of  the  different  breeds  under  comparative  feeding  conditions,  in  order 
to  obtain  a  correct  estimate  of  the  breed  relation. 

It  would  naturally  be  expected  that  the  most  favorable  condition 
for  studying  the  accuracy  of  the  views  held  by  the  cattle  breeders  and 
others  that  some  breeds  of  cows,  such  as  the  Jersey  and  Guernsey, 
are  color  producers  while  other  breeds,  such  as  the  Holstein,  are  not 
color  producers  would  be  a  comparison  of  the  color  of  the  combined 
fat  of  several  cows  of  each  breed.  Table  17,  which  follows,  gives 
such  a  comparison  taken  from  animals  in  one  herd.  The  milk  and 
fat  production  of  the  various  cows  varied  widely.  The  comparison 
was  made  during  the  winter  months,  the  only  source  of  pigment  being 
a  more  or  less  variable  quantity  of  green  alfalfa  hay  in  the  ration, 
which  was,  however,  the  same  for  all  the  animals. 


378    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 

TABLE  No.  17.     RELATION  OF  BREED  TO  COLOR  OF  MILK  FAT. 


Color 

of  butter  fc 

it 

Breed 

Yellow 

Red 

Light 

Jersey  

50.0 

2.1 

0.2 

Ayrshire  

38.0 

1.7 

0.2 

Shorthorn 

34  0 

1  6 

0.2 

Holstein 

31  0 

1.7 

0.2 

The  most  striking  fact  brought  out  by  this  table  is  that  the  ques- 
tion of  the  color  of  the  fat  produced  by  the  four  breeds  represented 
is  not  one  of  presence  or  absence  of  color,  but  rather  a  question  of 
relative  color.  The  fat  from  the  Jersey  cows  was  unquestionably  the 
highest  colored  of  the  four  samples  but  the  fat  from  the  Holsteins 
also  had  a  very  good  color,  although  the  butter  would  probably  have 
been  scored  as  "slightly  low  in  color." 

This  point  of  relative  color  production  is  also  clearly  shown 
when  comparing  the  fat  produced  by  individual  members  of  the  breeds. 
Table  18  shows  the  color  of  the  fat  from  two  Jerseys  and  one  Hol- 
stein cow  under  feeding  conditions  most  favorable  for  the  maximum 
color.  These  animals  were  producing  about  the  same  amount  of  but- 
ter fat,  and  the  roughage  of  their  ration  consisted  for  the  most  part 
of  freshly-cut  soybeans,  very  rich  in  carotin  and  xanthophylls. 


TABLE  No.  18. 


SHOWING  RELATIVE  COLOR  PRODUCTION  BY   DIFFERENT  IN- 
DIVIDUALS. 


Color 

Cow  No. 

Breed 

Feed 

Yellow 

Red 

Light 

59 

Jersey 

Fresh   green   soybeans 
and  grain 

54.0 

2.5 

1.0 

34 
208 

Jersey 
Holstein 

Ditto 
Ditto 

60.0 
29.0 

2.5 
1.8 

1.0 
0.5 

When  comparing  the  color  of  the  fat  produced  by  individual 
members  of  the  Jersey  and  Holstein  breeds  under  feeding  conditions 
favorable  for  only  a  moderate  amount  of  color  in  the  fat,  the  relative 
color  production  of  the  breeds  very  nearly  approaches  unity.  This  is 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT         379 


especially  true  when  the  fat  production  and  the  actual  proportion  of 
the  ration  furnishing  the  pigments  are  taken  into  account.  Such  a 
comparison  is  shown  in  Table  19. 

TABLE   No.    19.     RELATIVE   COLOR   PRODUCTION   UNDER   SPECIAL   FEEDING 

CONDITIONS. 


Cow 

No. 

Breed 

Pounds 
green 
alfalfa 
hay 

Green  feed 
(Dry 
matter 
basis)  (a) 

Pounds 
milk 
fat 
per  day 

Color 

Yellow 

Red 

34 
41 
11 
13 
208 
211 
219 

^  ersey 
'm  ersey 
^  ersey 
.  ersey 
iolstein 
Holstein 
Holstein 

10.0 
12.0 
8.0 
8.0 
15.0 
12.0 
12.0 

42% 
41% 
40% 
45.5% 
35% 
28% 
28% 

1.34 
1.78 
,      1.51 
0.58 
1.23 
2.13 
1.50 

29.0 
29.0 
24.0 
33.0 
19.0 
17.0 
19.0 

1.6 
1.6 
1.3 
1.6 
1.6 
1.5 
1.7 

(a)     Per  cent  of  moisture  free  green  feed  in  total  ration. 

The  most  interesting  feature  of  the  above  table  is  to  note  that 
on  the  basis  of  the  per  cent  of  green  dry  matter  in  the  ration,  the 
Jersey  cows  produced  the  highest  colored  fat  because  they  received 
the  highest  per  cent  of  green  dry  matter.  By  taking  into  considera- 
tion also  the  difference  in  fat  production,  an  interesting  calculation 
can  be  made  with  the  figures  of  average  color,  fat  production  and  per 
cent  of  green  dry  matter  in  the  ration,  which  will  cause  the  relative 
color  production  of  the  two  breeds  to  approach  almost  unity. 


Jerseys 

Holsteins 

Average  per  cent  of  green  feed  in  ration 

42  1% 

30% 

Average  fat  production 

1  301bs. 

1.  6  Ibs 

Average  color  of  fat  (Units  of  yellow) 

29  0 

18  0 

If  it  be  assumed  for  the  moment  that  there  is  no  breed  charac- 
teristic we  can  say  that  30%  green  feed  in  the  ration  of  the  Holsteins 
produces  18  units  of  yellow  in  the  fat  for  the  same  reason  that  42% 
green  feed  in  the  ration  of  the  Jerseys  produces  29  units  of  yellow 
in  their  fat.  If  this  is  true  then  the  following  proportion  would  be  a 
true  one,  i.  e. : 

42:29   :  :     30:18 

The  product  of  the  means  is  not  quite  equal  to  the  product  of  the 
extremes  but  gives  the  result, 

870  =  756 


380  MISSOURI  AGRICULTURAL  EXP.  STA.,  RESEARCH  BULLETIN  NO.  IO 


If  the  amount  of  fat  produced  is  taken  into  consideration  and  each  side 
of  this  equation  is  multiplied  by  the  corresponding  amount  of  fat  we 
have  the  result, 

870  X  1.3  =  756  X  1.6 
or  1,131  =  1,210 

or  i  =  1.07,  which  is  very  near  unity. 

The  relation  between  the  breed  of  the  cow  and  the  color  of  the 
fat  under  two  different  conditions  of  feeding  is  well  illustrated  by 
Tables  18  and  19.  The  color  of  the  fat  produced  by  cows  No..  34 
and  208  is  given  under  both  heavy  and  moderate  pigment  feeding. 
The  data  in  Table  19  were  obtained  a  number  of  weeks  after  that  in 
Table  18.  The  figures  show  that  the  change  from  heavy  to  moderate 
pigment  feeding  caused  the  color  of  the  milk  fat  of  the  Jersey  cow 
to  drop  50%  while  a  similar  change  in  the  feed  of  the  Hblstein  cow 
caused  a  color  drop  of  only  35%. 

The  relation  of  the  breed  to  the  change  in  color  produced  by  a 
change  in  the  ration  is  also  well  illustrated  in  the  following  table 
No.  20. 

TABLE  No.  20.     COLOR  PRODUCTION  IN  DIFFERENT  BREEDS  AS  AFFECTED  BY 

CHANGES  IN  RATION. 


Cow  No. 

Breed 

Date 

Grams 
fat 
produced 

Color  of  fat 

Yellow 

Red 

213 
213 

Holstein 
Holstein 

3-11-13 
4-10-13 

122 
135 

8.5 
54.0 

1.4 
1.8 

220 
220 

Holstein 
Holstein 

3-11-13 
4-10-13 

167 
208 

3.0 
22.0 

0.7 
1.2 

303 
303 

Ayrshire 
Ayrshire 

3-11-13 
4-10-13 

213 
263 

2.5 
16.0 

0.6 
1.1 

16 
16 

Jersey 
Jersey 

3-11-13 
4-10-13 

304 
363 

11.0 
64.0 

1.7 
2.0 

57 
57 

Jersey 
Jersey 

3-11-13 
4-10-13 

240 
263 

5.2 
54.0 

1.2 
1.7 

64 
64 

Jersey 
Jersey 

3-11-13 
4-10-13 

281 
358 

4.7 
47.0 

1.5 
1.6 

The  first  sample  for  each  cow  in  the  above  table  represents  the 
result  of  a  long  continued  feeding  of  a  ration  almost  entirely  lacking 
in  carotin  and  xanthophylls.  The  second  sample  represents  one  month's 
feeding  of  a  ration  rich  in  these  pigments,  the  ration  including  a 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF  MILK  FAT         381 

plentiful  supply  of  green  alfalfa  hay  and  some  fresh  green  grass. 
The  data  bring  out  two  points  worthy  of  emphasis.  One  of  these 
is  that  it  is  possible  to  find  pure  bred  Holstein  cows  entirely  lacking 
in  the  so-called  breed  characteristic  of  color  production.  Holstein 
Cow  No.  213  for  instance  produced  as  much  color  in  her  fat  in  both 
periods  as  any  of  the  Jerseys.  The  low  color  of  the  milk  fat  of  cows 
No.  220  and  No.  303  in  the  second  period  can  only  be  explained  at 
present  on  the  ground  that  it  was  due  to  some  inherent  characteristic 
of  the  animals,  which  for  lack  of  a  better  term  may  be  called  breed 
characteristic.  The  other  point  brought  out  by  the  data  is  merely  in 
emphasis  of  the  results  obtained  in  the  feeding  experiments  showing 
that  all  breeds  of  cows  suffer  alike  in  regard  to  the  color  of  their 
milk  fat  when  the  pigments  carotin  and  xanthophylls  are  withdrawn 
from  their  food.  At  this  time  there  is  no  breed  characteristic. 

Not  only  does  the  breed  characteristic  disappear  when  the  source 
of  the  pigment  is  withdrawn,  but  it  also  disappears  for  all  cows  at  the 
time  of  maximum  color  in  the  fat,  i.  e.  immediately  after  parturition. 
Data  was  given  in  Table  8  showing  the  high  color  of  the  colostrum 
milk  fat  for  cows  of  three  breeds.  There  was  certainly  no  breed 
characteristic  evident  there. 

There  is  one  other  breed  difference  yet  to  be  considered,  which 
has  led,  probably  more  than  anything  else,  to  the  belief  that  Jersey 
and  Guernsey  cows  can  produce  yellow  butter  fat  at  any  time,  regard- 
less of  feed.  This  difference  has  to  do  primarily  with  the  storage  of 
pigment  in  the  body,  and  its  discussion  belongs  properly  to  the  two 
subsequent  papers  of  this  series.  A  brief  statement  here  in  regard 
to  it  however  will  prevent  a  doubt  arising  in  the  minds  of  some  read- 
ers, whose  practical  experience  is  apparently  contrary  to  the  experi- 
mental evidence  here  offered. 

Stating  the  question  in  hypothetical  form,  it  may  be  said  that 
if  a  Jersey  (or  Guernsey)  and  a  Holstein  cow,  both  giving  well-colored 
milk  fat,  the  possibility  of  which  cannot  be  denied  in  the  light  of  the 
evidence  which  has  been  offered  on  this  point,  are  put  upon  dry  feed 
containing  little  or  no  carotin  and  xanthophylls,  the  color  of  the  milk 
fat  will  drop  much  faster  with  the  Holstein  cow  than  with  the  Jer- 
sey (or  Guernsey)  cow,  unless  great  care  is  taken  to  provide  a  ration 
as  nourishing  and  palatable  as  the  previous  pigmented  one.  The  re- 
sult will  be  that  the  Jersey  (or  Guernsey)  cow  will  appear  to  be  pro- 
ducing colored  milk  fat  on  a  non-pigmented  ration.  The  explanation 
for  this  has  already  been  given  in  connection  with  feeding  Experi- 
ments Nos.  i  and  6,  and  lies  in  the  fact  that  the  body  fat  of  Jersey 
and  Guernsey  cows  furnishes  a  supplementary  storage  of  pigments 


382    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH  BULLETIN   NO.    IO 

not  usually  found  in  other  breeds.  It  will  be  shown  in  a  subsequent 
paper  that  if  the  body  fat  which  furnishes  the  supplementary  pig- 
ments in  the  case  of  the  Jersey  (or  Guernsey)  cow  is  laid  on  with 
a  non-pigmented  ration,  it  will  be  as  colorless  as  is  often  seen  in  the 
case  of  the  body  fat  of  Holstein  cows.  If  this  were  true  in  the 
hypothetical  case  described  above,  there  would  have  been  no  breed 
characteristic  evident,  for  it  will  also  be  shown  in  a  subsequent  paper 
that  the  normal  storage  of  pigment,  i.  e.  that  of  the  blood  serum,  is 
practically  the  same  for  all  breeds  of  cows. 

Sufficient  evidence  has  been  presented  to  permit  a  repetition  of 
a  previous  statement,  namely  that  the  relation  of  the  breed  to  the 
color  of  the  milk  fat  has  received  more  emphasis  than  a  study  of  the 
question  will  warrant.  The  color  of  the  milk  fat  is  primarily  depend- 
ent upon  the  character  of  the  food  and  the  fact  that  some  breeds  of 
cows  give  less  color  in  their  milk  fat  than  other  breeds  will  probably 
be  found  to  be  only  an  apparent  one  when  all  the  factors  which  come 
into  play  are  known. 


CAROTIN,  THE  PRINCIPAL  \ELLOW   PIGMENT  OF  MILK  FAT         383 

DISCUSSION  OF  RESULTS. 

It  was  the  primary  object  of  this  investigation  to  classify  the 
natural  yellow  pigment  of  milk  fat  both  as  an  individual  and  also 
in  relation  to  the  two  well-known  yellow  classes  of  plant  pigments,  car- 
tin  and  xanthophylls,  whose  general  properties  have  often  been  observed 
to  be  closely  related  to  various  yellow  pigments  of  so-called  animal 
origin. 

Basing  the  study  upon  a  number  of  well-defined,  characteristic 
physical  and  chemical  properties  of  carotin  and  xanthophylls,  it  has 
been  shown  that  the  principal  pigment  of  milk  fat  is  a  member 
of  the  fast  widening  group  of  hydrocarbon  pigments,  the  carotin 
of  green  plants.  In  addition  it  has  been  shown  that  the  milk  fat 
carotin  nearly  always  has  associated  with  it  one  or  more  minor 
constituents  whose  general  properties  and  characteristics  are  identical 
with  the  xanthophyll  group  of  pigments.  Two  and  possibly  three 
xanthophyll  constituents  were  found  in  one  sample  of  high  colored 
butter  fat. 

In  addition  to  the  establishment  of  a  chemical  relation  between 
carotin  and  xanthophylls  and  the  yellow  lipochrome  of  milk  fat,  it 
has  been  possible  to  demonstrate  a  much  more  significant  fact,  namely 
that  this  lipochrome  whose  origin  has  hitherto  been  considered  to  be 
in  the  animal  body  is  in  reality  merely  the  carotin  and  xanthophylls 
of  the  food,  which  are  absorbed  by  the  body  and  subsequently  secreted 
in  the  milk  fat.  Numerous  feeding  experiments  show  that  when  the 
food  is  deficient  in  carotin  and  xanthophylls  for  a  period  of  time,  the 
milk  fat  slowly  decreases  in  color  and  eventually  approaches  a  color- 
less condition.  The  experiments  also  show  that  when  foods  rich  in 
carotin  and  xanthophylls  are  given  to  a  cow  whose  milk  fat  is  deficient 
in  lipochrome,  the  color  of  the  milk  fat  at  once  increases  in  propor- 
tion to  the  amount  of  pigments  fed.  This  is  true  regardless  of  whether 
the  carotins  and  xanthophylls  are  associated  with  chlorophyll  as  in 
green  feeds,  or  whether  chlorophyll  is  completely  absent  and  xan- 
thophylls almost  so,  as  in  carrots. 

The  experiments  show  in  addition  that  small  amounts  of  carotin, 
such  as  are  present  in  the  oil  of  cottonseed  meal  have  apparently  no 
effect  on  the  color  of  the  butter  fat.  It  is  not  clear,  however,  whether 
this  is  due  to  the  smallness  of  the  amount  of  carotin  or  to  the  state 
in  which  it  exists  in  the  food,  i.  e.,  dissolved  in  oil.  There  is  some 
evidence  on  both  sides.  Mendel  and  Daniels  *  have  recently  found 

1.     Jour.  Biol.  Chem.  13,  No.  1,  p.  72  (1912). 


384    MISSOURI  AGRICULTURAL  EXP.   STA.,   RESEARCH   BULLETIN   NO.    IO 

that  when  fifteen  grams  per  day  of  Sudan  III  dissolved  in  oil  was 
fed  to  a  cow  for  three  successive  days,  there  was  no  indication  of 
the  dye  in  the  milk.  On  the  other  hand,  in  the  feeding  experiments 
with  Cow  No.  301  where  the  bleached  alfalfa  hay  was  changed  to 
timothy  hay  containing  a  small  amount  of  carotin,  there  was  also 
apparently  no  effect  on  the  color  of  the  milk  fat. 

It  is  especially  noteworthy  that  all  of  the  above  feeding  experi- 
ments which  involved  yellow  corn  are  united  in  pointing  to  its  inabil- 
ity to  impart  any  color  to  the  butter  fat.  This  result  is  not  so  sur- 
prising, however,  when  viewed  in  the  light  of  the  character  of  the 
pigment  of  yellow  corn  as  shown  in  the  chemical  studies.  It  was 
found  there  that  the  pigment  is  largely  a  xanthophyll.  It  may  be 
stated  that  the  butter  fat  of  Cow  No.  301  during  the  last  yellow  corn 
experiment  failed  to  show  the  presence  of  the  corn  xanthophyll,  when 
subjected  to  careful  examination. 

The  feeding  of  carotin  in  the  form  of  carrots  to  a  cow  giving  as 
low  colored  milk  fat  as  Cow  No.  301  gave  an  excellent  opportunity  to 
study  the  proportion  of  carotin  and  xanthophyll  in  the  resulting  well- 
colored  butter  fat.  This  investigation  was  reported  in  connection 
with  the  study  of  the  proportion  of  carotin  and  xanthophyll  in  butter 
fat  under  varying  conditions  of  production.  It  was  found  that  the 
xanthophylls  were  practically  absent  from  the  fat.  The  conclusion 
is  that  the  xanthophylls  must  be  present  in  the  food  in  large  excess, 
as  in  grass,  before  they  will  appear  in  the  butter  fat. 

It  was  mentioned  above,  and  the  fact  is  worthy  of  special  notice, 
that  when  the  carotin  and  xanthophylls  are  withdrawn  from  the  food 
the  falling  off  in  color  of  the  butter  fat  is  sometimes  very  slow.  In  the 
case  of  Jersey  cow  No.  57,  it  required  twenty-seven  days  for  the  butter 
fat  to  drop  in  color  from  43  to  8.5  units  of  yellow.  Normally,  it  should 
require  much  less  time  for  the  color  to  drop  this  amount.  For  instance 
it  required  only  12  days  for  a  similar  drop  to  be  brought  about  in  the 
color  of  the  milk  fat  of  Jersey  cow  No.  59.  In  explanation  of  this 
difference  it  may  be  stated  that  upon  a  normal  plane  of  nutrition,  the 
blood  serum  furnishes  the  pigment  for  the  milk  fat.  When  the  plane 
of  nutrition  is  below  normal  in  a  lactating  cow  the  body  fat  is  drawn 
upon  to  aid  in  the  production  of  milk  fat  and  also  for  other  purposes. 
If  the  body  fat  thus  utilized  has  a  high  yellow  color,  as  is  usually 
the  case  in  Jersey  and  Guernsey  cows,  the  normal  storage  of  pigment 
for  the  milk  fat  will  be  continually,  at  least  partially,  replenished. 
The  reduction  in  color  of  the  milk  fat  will  then  be  much  slower  than 
normal.  It  was  stated  above  and  will  bear  repetition,  that  we  have 
here  an  explanation  of  why  Jersey  cows  apparently  often  produce  high 
colored  milk  fat  on  a  low  pigmented  ration,  as  during  the  winter  months. 


CAROTIN,  THE  PRINCIPAL   YELLOW   PIGMENT  OF  MILK   FAT         385 

The  results  of  these  experiments  are  of  considerable  practical 
importance.  It  is  readily  seen,  for  instance,  that  the  peculiar  popular 
conception  of  the  enhanced  value  of  butter  on  account  of  a  high 
yellow  color  is  absolutely  without  foundation.  It  is  furthermore  seen 
that  the  prevailing  opinion  among  some  cattle  breeders  that  Guernsey 
and  Jersey  cows  are  able  to  synthetically  produce  a  high  colored  but- 
ter fat  under  all  conditions  is  also  unfounded.  It  has  been  shown 
that  all  breeds  of  cows  will  produce  well-colored  butter  fat  under 
proper  feeding  conditions.  The  reverse  has  been  shown  to  be  espe- 
cially true,  namely  that  a  cow,  regardless  of  breed,  cannot  produce 
high  colored  butter  fat  under  normal  conditions,  unless  the  food  con- 
tains the  pigments  which  are  utilized  for  that  purpose. 

With  our  present  knowledge,  however,  we  would  not  be  justified 
in  saying  that  there  is  no  breed  characteristic  in  connection  with  the 
color  of  butter  fat.  Under  apparently  equal  conditions  Jersey  and 
Guernsey  cows  usually  give  higher  colored  milk  fat  than  Holstein  or 
Ayrshire  cows.  We  have  been  able  to  offer  some  evidence,  however, 
showing  that  during  a  moderate  pigmentation  of  the  milk  fat,  this 
difference  largely  disappears  when  the  amount  of  fat  produced  and 
the  proportion  of  the  ration  which  is  the  source  of  the  pigment  are 
taken  into  consideration.  Further  experiments  would  be  required  to 
ascertain  whether  this  is  true  under  all  conditions  of  milk  fat  pig- 
mentation or  only  true  for  moderate  pigmentation.  The  data  already 
at  hand  at  least  entirely  justify  the  statement  that  the  so-called  breed 
characteristic  has  been  given  more  emphasis  than  is  warranted  by  an 
actual  study  of  the  facts. 

The  results  of  our  experiments  are  furthermore  of  considerable 
physiological  significance.  A  direct  source  for  the  lipochromes  of  the 
cow  has  been  established,  which  opens  the  question  of  a  similar 
source  for  all  animal  lipochromes.  The  lipochrome  of  milk  fat  has 
been  increased  or  decreased  with  great  ease  by  merely  varying  the 
food  of  the  cow.  Such  a  result  throws  great  doubt  upon  any  physio- 
logical significance  which  the  lipochromes  have  been  supposed  to  exert 
in  the  animals  in  which  they  have  been  found.  Apparently  it  is  merely 
a  question  of  the  inability  of  the  animal  body  to  throw  off  the  excess 
of  carotin  and  xanthophylls  contained  in  its  food.  Experiments  will 
reported  in  a  later  paper  of  this  series  showing  that  the  blood  serum, 
of  the  cow  at  least,  very  rapidly  takes  up  the  carotin  (especially)  of 
the  food  and  carries  it  through  the  body  in  combination  with  an  albu- 
min of  the  serum. 


386    MISSOURI  AGRICULTURAL  EXP.   STAV   RESEARCH   BULLETIN   NO.    IO 


SUMMARY. 

1.  The  fat  of  cows'  milk  owes  its  natural  yellow  color  to  the 
pigments  carotin  and  xanthophylls,  principally  carotin,  the  well-known, 
wide-spread,  yellow  vegetable  pigments  found  accompanying  chloro- 
phyll in  all  green  plants. 

2.  The  carotin  and  xanthophylls  of  milk  fat  are  not  synthesized 
in  the  cow's  body,  but  are  merely  taken  up  from  the  food  and  subse- 
quently secreted  in  the  milk  fat. 

3.  When  food  practically  free  from  carotin  and  xanthophylls, 
such  as  the  cow  usually  receives  during  the  winter  months,  is  given 
to  a  milk-giving  cow,  the  immediate  supply  of  these  pigments  in  the 
organism  is  greatly  depleted  and  may  be  entirely  used  up,  on  account 
of  the  constant  drain  upon  the  supply  by  the  milk  glands.     The  but- 
ter fat  accordingly  approaches  a  colorless  condition  in  proportion  to 
the  supply  of  carotin  and  xanthophylls  in  the  system,  the  length  of 
time  these  pigments  are  kept  out  of  the  food,  and  also,  very  probably, 
in  proportion  to  the  amount  of  milk  fat  being  produced. 

4.  If  food  rich  in  carotin  and  xanthophylls  is  given  to  a  milk- 
giving  cow  whose  milk  fat  has  become  practically  colorless  by  reason 
of  the  above  conditions,  the  organism  will  at  once  recover  its  lost 
pigments  and  the  milk  fat  will  increase  in  color  in  proportion  to  the 
amount  of  carotin  and  xanthophylls,  especially  carotin,  in  the  food. 
Fresh  green  grass  probably  being  the  richest  in  carotin  of  all  natural 
dairy  cattle  feeds,  accordingly  produces  the  highest  colored  butter. 

5.  There  is   some  difference  among  different  breeds  of  dairy 
cows  in  respect  to  the  maximum  color  of  the  milk  fat  under  equally 
favorable  conditions  for  the  production  of  a  high  color.     Each  breed 
of  cows,  however,  will  undergo  the  same  variation  in  color  of  the 
milk  fat  which  follows  a  withdrawal  or  addition  of  carotin  and  xan- 
thophylls, especially  carotin,  to  the   food.     Under   some  conditions, 
also,  the  apparent  breed  characteristic  largely  disappears.     The  popu- 
lar opinion  in  regard  to  the  breed  characteristic  has  been  overempha- 
sized, and  statements  in  regard  to  it  should  in  the  future  be  qualified 
with  a  statement  of  the  conditions  of  feed,  etc. 

6.  Under  normal  conditions  cows  of  all  breeds  produce  very  high 
colored  milk  fat  for  a  short  time  after  parturition.     The  pigments 
of  the  fat  at  this  time  are  identical  with  the  normal  pigments  of  the 
fat.     Their  increase  at  this  time  is  probably  due  to  the  physiological 
conditions  surrounding  the  secretion  of  the  milk  of  the  freshening 
animal. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF  MILK  FAT         387 


BIBLIOGRAPHY. 

1.  Escher:    Zeit.  f.  Physiol.  Chem.  83,  p.  198  (1913). 

2.  Kirsten:    Zeit.  Nahr.  Genussm.  5,  p.  833  (1902). 

3.  Kraus:     Flora,  p.  155  (1875). 

4.  Lewkowitsch:    "Oils.  Fats  and  Waxes,"  vol.  i,  p.  371  (1909  Edi- 
tion) . 

5.  Mendel  and  Daniels:    Jour.  Biol.  Chem.  13,  No.  i,  p.  72    (1912). 

6.  Newbigin:     D.  Noel  Paton — "Reports  of  Investigations  on  Life 
History  of  Salmon"  (1898),  Article  XV,  p.  159. 

7.  Schunck:    Proc.  Roy.  Soc.  72  (1903). 

8.  Sorby:     Proc.  Roy.  Soc.  21,  p.  456    (1875). 

9.  Thudichum:    Proc.  Roy.  Soc.  17,  p.  253  (1869). 

10.    Tswett:    Ber.  d.  Deut.  Botan.  Gessel.  24,  pp.  316  and  384   (1906)  ; 

29,  p.  630    (1911). 
n.    Willstatter  and  Escher:     Zeit.  f.  Physiol.  Chem.  76,  pp.  214-225 

(1912). 

12.  Willstatter  and  Mieg:    Ann.  d.  Chemie  355,  p.  i    (1907). 

13.  Windaus:    Zeit.  f.  Physiol.  Chem.  65,  p.  no    (1909). 


CAROTIN— THE  PRINCIPAL  NATURAL  YELLOW 
PIGMENT  OF  MILK  FAT.*— PART  III.  ;, 


The  Figments  of  the  Body  Fat,  Corpus  Luteum  and  Skin  Secretions 

of  the  Cow. 

LEROY  S.  PALMER  AND  C.  H.  ECKLES 

Recent  investigations  in  regard  to  yellow  animal  pigments  have 
shown  that  some  of  them  are  closely  related  chemically  or  identical 
with  yellow  pigments  of  plant  origin.  Willstatter  and  Escher  *  have 
found  that  the  pigment  of  egg  yolk  is  isomeric  with  a  crystalline  xan- 
thophyll  of  green  plants,  and  Escher2  has  found  the  pigment  of  the 
corpus  luteum  to  be  identical  with  the  widely  distributed  hydro- 
carbon pigment,  the  carotin  of  fruits,  flowers  and  green  plants. 

In  a  study  of  the  commonly  observed  yellow  lipochrome  of  but- 
ter fat  we  have  found3  that  it  is  composed  principally  of  a  pigment 
identical  with  carotin,  with  one  or  more  minor  constituents  which 
are  evidently  identical  with  the  xanthophyll  pigments.  We  have  fur- 
thermore shown  by  an  unbroken  chain  of  evidence  that  these  pigments 
are  present  in  the  milk  fat  as  a  result  of  feeding  a  ration  containing 
an  abundant  amount  of  these  pigments.  The  presence  of  these  pig- 
ments in  milk  fat  is  therefore  not  due  to  any  synthetic  powers  which 
the  animal  possesses,  but  merely  to  the  fact  that  the  organism  ab- 
sorbs the  pigments  along  with  the  products  of  food  digestion  and 
subsequently  secretes  them  dissolved  in  the  milk  fat.  We  were  ac- 
cordingly able  to  vary  the  amount  of  pigment  in  the  milk  fat  by  sim- 
ply choosing  the  proper  feeds,  i.  e.,  either  deficient  in  carotin  and 
xanthophylls  or  very  rich  in  these  pigments.  The  above  relations 
between  the  carotin  and  xanthophylls  of  milk  fat  and  the  carotin 
and  xanthophylls  of  feeds  were  found  to  hold  good  for  all  breeds 
of  dairy  cows. 

1.  Zeit.  f.  Physiol.  Chem.  76,  pp.  214-225  (1912). 

2.  Zeit.  f.Physiol.  Chem.  83,  p.  198  (1913. 

3.  Research  Bulletin  No.  10   Missouri  Agr.  Exp.  Sta;  Jour.  Biol.  Chem. 
17,  p.  191  (1914). 

*See  Foreword,  Part  I,  for  statement  of  co-operation  with  Dairy  Division, 
U.  S.  Department  of  Agriculture. 

(391) 


392  MISSOURI  AGRICULTURAL  EXP.   STA.,  RESEARCH  BULLETIN  NO.   II 

The  establishment  of  the  chemical  identity  of  the  pigment  of 
milk  fat  and  of  its  simple  physiological  relation  to  the  carotin  and 
xanthophylls  of  green  plants  at  once  opened  the  question  of  a  sim- 
ilar relation  of  the  pigment  which  is  so  often  observed  in  the  body 
fat  of  cows,  especially  those  of  certain  breeds,  such  as  the  Jersey  and 
Guernsey.  The  question  is  also  raised  as  to  the  presence  of  xan- 
thophylls in  the  corpus  luteum  pigment.  In  addition  an  interesting 
question  is  opened  as  to  whether  the  yellow  skin  secretions  of  cer- 
tain breeds  of  dairy  cows,  which  is  often  interpreted  as  indicating 
the  ability  of  these  animals  to  secrete  yellow  milk  fat,  is  also  due 
to  the  same  pigments  that  characterize  the  butter  fat. 

The  present  investigation  was  undertaken  for  the  purpose  of 
studying  these  questions.  In  addition  some  information  was  gathered 
relative  to  the  relation  of  the  breed  of  the  cow  to  the  amount  of  color 
found  in  the  body  fat. 

METHODS  OF   IDENTIFICATION. 

The  general  methods  of  studying  and  identifying  the  pigments 
of  the  body  fat,  corpus  luteum  and  skin  secretions  of  the  cow  were 
the  same  as  were  used  in  the  study  of  the  pigment  of  milk  fat..  A 
detailed  account  of  these  methods  may  be  found  in  the  preceding 
paper  of  this  series,  which  deals  with  the  milk  fat  pigment. 

These  methods  were  a  study  of  what  we  have  called  the  spec- 
troscopic,  solubility,  and  adsorption  properties  of  the  pigments.  The 
methods  were  confined  to  characteristic,  physical  and  chemical  prop- 
erties of  the  pigments  for  the  same  reasons  that  they  were  used  for 
the  study  of  the  milk  fat  pigment,  namely  because,  in  the  case  of  the 
body  fat  at  least,  the  very  large  amount  of  fat  with  which  the  pig- 
ments are  associated  precludes  their  isolation  in  sufficient  quantity 
for  chemical  analysis.  In  the  case  of  the  pigments  of  the  corpus 
luteum  and  skin  secretions  not  enough  material  was  available  for 
isolating  any  great  quantity  of  pigment.1 

The  Spectroscopic  Properties. — It  was  found  that  carotin  and 
xanthophylls  isolated  from  green  alfalfa  hay,  carrots  or  other  plants 
rich  in  these  pigments  showed  characteristic  absorption  bands  when 
viewed  in  a  spectroscope  of  narrow  dispersion.  When  the  spectro- 
scope was  set  at  an  arbitrarily  chosen  standard,  each  class  of  pig- 
ments exhibited  bands  in  characteristic  position,  especially  in  carbon 
bisulphide  solution,  and  could  then  be  readily  identified.  The  arbitrary 

1.  Escher  succeeded  in  isolating  less  than  0.5  gram  of  impure  crystals  of 
corpus  luteum  pigment  from  10,000  cows'  ovaries. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK   FAT         393 

standard  was  obtained  by  fixing  the  arbitrary  scale  attached  to  the 
spectroscope  at  a  constant  figure  which  was  furnished  by  a  sodium 
flame,  the  spectrometer  slit  being  closed  to  furnish  the  narrowest 
possible  line.  This  standard  did  not  of  course  give  absolute  meas- 
urements of  absorption  bands  but  merely  a  means  of  comparing  the 
position  of  bands  of  various  solutions,  which  was  the  desired  end 
in  view.  Before  measuring  the  bands  of  an  unknown  pigment,  the 
strength  of  its  solution  was  adjusted  to  give  bands  of  as  nearly  the 
same  intensity  and  clearness  as  the  bands  whose  arbitrary  measure- 
ments furnished  the  standard.  The  arbitrary  standards  for  the  ab- 
sorption bands  of  carotin  and  xanthophylls,  which  were  adopted,  are 
given  in  Table  I. 


TABLE  1. — SPIJCTROSCOPIC  STANDARDS  or  CAROTIN  AND  XANTHOPHYIXS. 


Pigment 

Solvent 

Measurement  of  absorption  bands. 

Band  I 

Band  II 

Band  III 

Carotin 
Carotin 

CS, 

C2H5(OH) 

225—242 
257—275 

261—278 
303—318 

301—319 
345—364 

Xanthophyll 
Xanthophyll 

CS2 

C2H5(OH) 

233—253 
263—280 

272—291 
305—325 

312—330 

355—..  . 

The  Solubility  Properties. — The  relative  solubility  properties  of 
carotin  and  xanthophylls  are  based  on  the  fact  that  organic  compounds 
are  best  soluble  in  solvents  of  similar  composition.  Accordingly  car- 
otin, which  is  a  hydrocarbon,  is  much  more  soluble  in  hydrocarbons 
like  petroleum  ether  than  in  the  alcohols.  Similarly  the  xanthophylls 
are  much  more  soluble  in  the  alcohols  than  in  a  hydrocarbon  like 
petroleum  ether.  These  phenomena,  as  stated  in  the  preceding  paper 
of  this  series,  were  discovered  and  elaborated  by  Tswett l  and  by 
Willstatter  and  Mieg.2  At  this  station  they  were  found  to  be  very 
characteristic  of  carotin  and  xanthophylls  and  in  addition  very  use- 
ful for  separating  and  differentiating  the  carotin  and  xanthophyll 
constituents,  not  only  of  plants  but  also  of  the  milk  fat  pigment. 
Thus  the  xanthophyll  constituents  of  a  mixed  pigment  could  be 
readily  separated  by  shaking  the  petroleum  ether  solution  of  the 

1.  Ber.  d.  Deut.  Botan.  Gessel.  24,  pp.  316,  384  (1906);  29,  p.  630  (1911). 

2.  Ann.  d.  Chemie.  355,  p.  1   (1907). 


394  MISSOURI  AGRICULTURAL  EXP.  STA._,  RESEARCH  BULLETIN  NO.   II 

mixed  pigment  with  eighty  to  ninety  per  cent  alcohol.  Or  if  an 
eighty  to  ninety  per  cent  alcoholic  solution  of  the  mixed  pigments 
was  shaken  with  petroleum  ether,  the  latter  solvent  would  completely 
extract  the  carotin,  leaving  the  xanthophylls  in  the  alcohol.  By  the 
second  method  especially,  it  was  possible  to  show  the  presence  of 
xanthophyll  pigments  in  butter  fat  which  could  not  be  extracted 
from  their  alcoholic  solution  by  petroleum  ether. 

The  Adsorption  Properties. — These  properties  were  discovered  by 
Tswett.1  They  are  based  on  the  fact  that  carotin  and  the  xan- 
thophylls show  a  great  difference  in  regard  to  the  ease  with  which 
they  enter  into  combination  with  certain  finely  divided  organic  and 
inorganic  compounds,  such  as  Inulin,  Saccharose  or  CaCO3  For 
instance,  carotin  is  not  adsorbed  at  all  by  CaCO3  from  its  perfectly 
anhydrous  carbon  bisulphide  or  petroleum  ether  solutions,  while  the 
xanthophylls  are  adsorbed  to  a  greater  or  kss  extent.  Briefly  then, 
it  has  been  found  that  if  a  carbon  bisulphide  solution  (in  which 
the  pigments  have  an  unusually  brilliant  color)  of  the  mixed  pig- 
ments is  filtered  slowly  through  a  column  of  CaCO3,  previously  mois- 
tened with  the  solvent,  the  pigments  will  be  differentiated  into  zone- 
like  rings  as  they  pass  through  the  column,  depending  on  their  ad- 
sorption affinity  towards  the  CaCO3.  Carotin  being  unadsorbed  will 
pass  through  first  as  a  rose  or  red  orange  colored  zone,  with  the 
various  xanthophylls  distributed  above  as  zones  of  different  shades 
of  yellow  or  orange.  The  xanthophylls  which  are  completely  ad- 
sorbed by  the  CaCOs  can  be  washed  out  afterwards  by  a  stream  of 
petroleum  ether  containing  ten  per  cent  absolute  alcohol. 

THE   PIGMENTS  OF  THE  BODY  FAT. 

The  pigment  of  the  body  fat  of  the  cow  has  never  been  subjected 
to  a  critical  examination.  Newbigin 2  reports  the  only  attempt  to 
identify  it.  He  extracted  the  pigment  from  a  sample  of  bright 
yellow  body  fat  and  compared  its  properties  with  those  of  a  yellow 
pigment  which  he  isolated  from  the  salmon,  which  pigment,  he  says, 
belongs  to  a  widely  distributed  group  of  animal  pigments  commonly 
confounded  with  the  lipochrome  pigments.  He  found  the  body  fat 
pigment  very  similar  in  properties  to  the  yellow  non-lipochrome 
pigment.  It  did  not  gfive  the  lipochrome  properties  and  was  very 
little  soluble  in  methyl  alcohol.  Newbigin  also  compared  the  body 

1.  Ber.  d.  Deut.  Botan.  Gessel.  24,  pp.  316,  384  (1906). 

2.  D.  Noel  Patton,  Report  of  Inv.  on    Life    History    of    Salmon    (1898), 
Article  XN,  p.  159. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT         395 

fat  pigment  with  the  pigment  from  maize,  with  the  result  that,  "The 
maize  pigment  gave  the  lipochrome  reaction  faintly  with  H2SO4, 
distinctly  with  HNO3  while  the  fat  pigment  gave  no  lipochrome  re- 
action. In  other  respects,  in  tint,  solubility,  etc.,  the  pigments  closely 
resembled  each  other."  The  experience  of  this  station  in  studying 
the  lipochrome  properties  of  the  milk  fat  pigment  seems  to  indi- 
cate that  Newbigin's  results  were  due  to  the  fact  that  he  was  work- 
ing with  decomposed  pigment.  Nevertheless  particular  attention  was 
paid  to  the  lipochrome  properties  of  the  body  fat  pigment. 

Method  of  Isolation. 

The  method  was  the  same  as  that  used  for  isolating  the  milk 
fat  pigment.  It  consisted  in  careful  saponification  of  the  fat  with 
alcoholic  potash  (2  c.c.  of  20%  solution  per  gram  of  fat)  and  sub- 
sequent extraction  of  the  unsaponifiable  matter  from  the  diluted 
soap  (3  volumes  of  water  to  one  of  soap)  with  ether.  The  ether  ex- 
tract was  sometimes  purified  by  re-saponification  and  re-extraction 
with  ether,  and  sometimes  was  freed  from  cholesterol  with  digitonin. 
Only  small  amounts  of  fat  were  used  for  each  test,  i.  e.,  25  to  30 
grams,  as  the  studies  of  the  milk  fat  pigment  showed  that  the  best 
results  could  thus  be  obtained. 

Identification  of  Pigments. 

Only  two  typical  experiments  showing  the  character  of  the  body 
fat  pigments  will  be  reported. 

Experiment  I. 

Twenty-five  to  thirty  grams  of  pure  rendered  kidney  fat  from 
a  Jersey  cow  was  used  for  this  experiment.  The  fat  had  a  high 
yellow  color  testing  in  the  one-inch  tintometer  cell  54  yellow,  2.3  red. 
The  unsaponifiable  matter  of  the  fat  had  a  golden  yellow  color  in 
ether  solution  and  a  blood  red  color  in  carbon  bisulphide  solution. 
The  carbon  bisulphide  solution  left  no  adsorbed  zone  in  the  CaCO3 
when  analyzed  chromotographically,  but  passed  through  unadsorbed 
as  an  orange-red  or  rose  colored  zone.  A  very  small  amount  of 
pigment  was  left  in  the  column,  however,  which  was  readily  washed 
out  by  a  stream  of  alcoholic  petroleum  ether. 

The  main  pigment  was  now  examined  with  respect  to  its  solu- 
bility relations  toward  petroleum  ether  and  80  per  cent  alcohol.  A 
very  minor  constituent  was  thus  obtained  more  soluble  in  80  per 


396  MISSOURI  AGRICULTURAL  EXP.  STAV  RESEARCH  BULLETIN  NO.    II 

cent  alcohol  than  in  petroleum  ether  (b.  p.  30-50).  This  constituent, 
when  combined  with  the  pigment  adsorbed  by  the  CaCO3  in  the 
chromotogramm,  did  not  show  sufficiently  sharp  absorption  bands 
for  accurate  measurements. 

The  main  petroleum  ether  soluble  pigment  was  transferred  to 
carbon  bisulphide  in  which  it  showed  two  strong  bands  and  a  third 
faint  one,  the  measurements  of  which  are  given  in  Table  2.  The 
residue  from  this  solution  gave  a  greenish-blue  color  with  concen- 
trated sulphuric  acid. 

,  Experiment  II. 

An  unusually  high  colored  fat  taken  from  the  back  of  a  Jersey 
cow  was  used  for  this  experiment.  The  pure  rendered  fat  tested 
in  the  one-inch  tintometer  cell  80  yellow,  2.7  red.  Thirty  grams 
of  this  fat  was  saponified  with  300  c.  c.  of  5  per  cent  alcoholic  pot- 
ash by  dissolving  the  fat  in  the  hot  alkaline  solution,  letting  stand 
for  24  hours  in  the  cold  with  frequent  shaking  and  finally  boiling  on 
the  steam  bath  for  one  half  hour.  By  using  this  procedure  not  a 
trace  of  foreign  pigment  was  developed.  One  extraction  with  ether 
rendered  the  diluted  soap  colorless.  The  golden  yellow  ether  ex- 
tract, after  purification,  was  concentrated  into  50  c.  c.  of  absolute 
alcohol.  On  careful  analysis  of  this  solution  it  was  found  possible 
to  separate  its  pigment  into  a  major  pigment  which  was  readily  ex- 
tracted by  petroleum  ether  and  a  minor  pigment  which  could  not  be 
extracted  by  petroleum  ether.  The  main  petroleum  ether  soluble 
pigment  was  readily  soluble  in  carbon  bisulphide  with  a  blood  red 
color,  and  in  this  solvent  showed  two  strong  absorption  bands  and 
a  third  faint  one,  the  measurements  of  which  are  given  in  Table  2. 
Analyzed  chromotographically,  the  carbon  bisulphide  solution  passed 
through  as  an  unadsorbed  beautiful  rose  colored  zone.  There  was 
no  differentiation.  The  residue  from  the  solution  gave  a  deep  blue 
color  with  concentrated  sulphuric  acid. 

The  alcohol  soluble  pigment,  which  probably  comprised  several 
per  cent  of  the  total,  was  transferred  to  ether  by  diluting  the  alco- 
holic solution  with  much  water  in  a  separatory  funnel.  Petroleum 
ether  was  added,  precipitating  some  water,  and  the  ethereal  solu- 
tion washed  with  water  until  clear.  The  solution  was  now  evap- 
orated and  the  yellow  residue  dissolved  in  carbon  bisulphide,  giving 
a  yellow-orange  solution  which  showed  two  fine  absorption  bands,  and 
a  third  fainter  one,  the  measurements  of  which  are  given  in  Table 
2.  The  bands  seem  to  be  shifted  more  toward  the  blue  than  the 
usual  xanthophyll  bands. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         397 


The  orange-yellow  carbon  bisulphide  solution  was  now  analyzed 
chromotographically.  Only  one  pigment  was  present,  which  passed 
through  the  column  very  slowly  as  a  narrow  orange  zone,  leaving 
no  pigment  in  the  CaCO3  which  could  be  washed  out  with  alcoholic 
petroleum  ether. 

TABLE  2. — ABSORPTION  BANDS  OF  CAROTIN  AND  XANTHOPHTTLLS  OF  BODY  FAT. 


Experiment 
No. 

Measurements  of  absorption  bands 

Carotin 

Xanthophylls 

1. 

Band  I 
Band  II 
Band  III 

223—242 
259—279 
300—319 

2. 

Band  I 
Band  II 
Band  III 

224—243 
262—288 
302—322 

Band  I                             235—252 
Band  II                            278—302 
Band  III                          315—335 

It  must  necessarily  be  concluded  from  these  experiments  that 
Newbigin's  "inert"  class  of  lipochromes  does  not  exist  in  the  body 
fat  of  the  cow,  but,  on  the  other  hand,  the  pigment  of  this  fat  is,  like 
the  butter  fat  pigment,  composed  of  a  major  carotin  and  one  or  more 
minor  xanthophyll  constituents,  all  of  which  also  show  the  proper- 
ties of  lipochromes.  It  is  to  be  noted  also  that  the  number  of  xan- 
thophylls  in  the  body  fat  varies,  as  was  found  to  be  the  case  in  the 
butter  fat  studies. 


The  Relation  Between  the  Cofor  of  the  Body  Fat  and  the  Food  of  the  Cow. 

Numerous  feeding  experiments  in  connection  with  the  study 
of  the  pigment  of  milk,  reported  in  the  preceding  paper  of  this  series, 
showed  that  the  carotin  and  xanthophylls  which  were  found  to  char- 
acterize the  milk  fat  were  present  there  on  account  of  the  fact 
that  the  food  contained  these  pigments.  Since  the  pigment  of  the 
body  fat  is  also  composed  of  carotin  and  xanthophylls  it  is  natural 
to  suppose  that  it  is,  like  the  pigment  of  milk  fat,  derived  from  the 
food,  the  carotin  and  xanthophylls  being  carried  to  the  fat  depots  and 
fat  synthesizing  body  cells  in  the  same  manner  that  they  are  carried  to 
the  milk  glands. 

In  order  to  obtain  evidence  of  this  fact,  however,  the  following 
experiment  was  undertaken.  Two  barren  and  dry  Jersey  cows  in 


398  MISSOURI  AGRICULTURAL  EXP.  STA.,  RESEARCH  BULLETIN  NO.   II 


moderate  flesh  were  fed  wheat  straw  alone  for  sixty  days  or  until  the 
animals  had  lost  as  much  fat  as  was  considered  necessary  for  the 
second  part  of  the  experiment.  The  daily  ration  of  cow  No.  25 
was  then  changed  to  9  pounds  of  yellow  corn  and  20  pounds  of  green 
alfalfa  hay,  which  was  rich  in  carotin  and  xanthophylls.  Cow  No.  21 
was  given  a  daily  ration  averaging  11.4  pounds  of  white  corn  and 
14  pounds  of  bleached  clover  hay,  very  deficient  in  carotin  and  xan- 
thophylls. Cow  No.  25  was  slaughtered  at  the  end  of  81  days.  Her 
gain  in  weight  during  this  period  was  160  pounds.  Cow  No.  21 
was  slaughtered  at  the  end  of  95  days.  She  had  gained  materially 
in  condition  during  her  "fattening"  period  although  the  scales  showed 
little  gain  in  weight.  This  was  probably  due  to  a  much  greater  "fill" 
when  receiving  wheat  straw.  Samples  of  fat  from  various  parts  of 
the  body  were  taken  from  each  cow  at  slaughtering  and  used  for 
color  readings.  The  results  aire  given  in  Table  3.  The  colorimetric 
readings  in  this  and  subsequent  tables  were  made  on  the  rendered, 
melted  fat,  measured  by  the  Lovibond  Tintometer.  A  complete  de- 
scription of  this  instrument  may  be  found  in  the  preceding  paper  of 
this  series. 

TABLE  3. — THE  RELATION  OF  FEED  TO  COLOR  OF  BODY  FAT. 


Color 

of  fat. 

Part  of  body. 

< 

:ow  No 

.  25. 

Cow  I 

^o.  21. 

Yellow 

Red 

Light 

Yellow 

Red 

Light 

Rib  plate  .           

50 

2.3 

1.0 

1.4 

0.1 

0 

Caul.                  

47 

2.1 

1.0 

3.6 

0.5 

0 

29 

1.3 

1.0 

8.0 

1.0 

0 

Around  ovaries  and  uterus 

49 

2.3 

1.0 

2.5 

0.3 

0 

Attached  to  fourth  stomach  .... 

33 

1.6 

1.0 

24.0 

1.7 

.0 

In  pelvic  cavity                               .  . 

50 

2.3 

1.0 

47,0 

2.1 

.0 

Kidney                                    

54 

1.6 

1.0 

50.0 

2.1 

.0 

Crops                           .          

50 

2.3 

1.0 

47.0 

1.9 

.0 

Over  last  rib             

47 

2.3 

1.0 

50.0 

2.1 

.0 

Over  outside  chuck.  . 

47 

2.0 

1.0 

47.0 

1.8 

.0 

The  results  of  this  experiment  are  even  more  striking  when 
the  amount  of  fat  on  the  various  parts  of  the  bodies  of  the  two 
cows  is  taken  into  consideration.  Aside  from  the  kidney  fat  and 
pelvic  cavity  fat,  which  were  probably  not  disturbed  to  any  extent 
during  the  starvation  period,  and  which  furthermore  were  of  equal 
color  in  the  two  animals,  the  largest  proportion  of  the  entire  fat 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK   FAT         399 

of  the  two  cows  was  represented  by  the  caul  fat.  The  caul  apron 
of  Cow  25  had  a  net  weight  of  12,132  grams;  that  of  Cow  21  a  net 
weight  of  7,364  grams.  It  is  here  that  the  great  difference  in  color 
is  noticeable.  In  fact,  if  all  the  fat  on  the  body  of  Cow  21  had 
been  rendered  and  its  color  compared  with  the  same  from  Cow  25, 
the  difference  in  color  would  have  been  very  marked. 

There  can  be  no  doubt  that  the  above  data  is  conclusive  as  to 
the  effect  of  feeding  a  non-pigmented  ration  to  a  fattening  cow  whose 
fat  had  been  largely  eliminated  by  starvation  previous  to  the  feed- 
ing of  the  colorless  ration.  In  other  words,  it  is  apparent  that  the 
body  fat  of  the  Jersey  cow  is  colored  primarily  because  the  food  is 
rich  in  carotin  and  xanthophylls  during  the  time  the  fat  is  on.  The 
blood  serum  of  both  cows  contained  carotin  at  the  time  of  slaugh- 
tering. Unfortunately  no  comparison  was  made  of  the  amount  in 
the  serum  of  the  two  cows.  No  doubt  there  was  a  much  smaller 
amount  in  the  blood  serum  of  Cow  21,  than  in  the  serum  of  Cow  25.  In 
both  cows  there  was  no  known  path  of  elimination  of  the  blood  pig- 
ment during  the  starvation  period,  the  cows  being  both  dry  and 
barren.  During  the  fattening  period  of  Cow  25,  the  demands  made 
on  the  blood  store  by  the  body  fat  producing  cells,  was  replenished 
by  the  food.  In  the  case  of  Cow  21,  however,  there  was  no  replen- 
ishing source,  and  the  amount  in  the  serum  must  have  greatly 
diminished. 

The  data  resulting  from  this  experiment  have  some  significance 
aside  from  the  relation  of  the  body  fat  to  the  food  of  the  cow.  The 
fact  that  the  outside  fats  of  the  two  cows  were  of  equal  color,  and 
the  inside  fats,  especially  the  caul  and  rib  plate  fat,  were  of  such 
widely  different  colors,  would  seem  to  indicate  what  fats  are  drawn 
upon  first  in  starvation  in  this  class  of  animals,  and  what  fats  are 
laid  on  first  during  fattening. 

Relation  Between  Color  of  Body  Fat  and  Breed  of  Cow. 

Considerable  attention  was  given  in  connection  with  the  study 
of  the  milk  fat  pigment,  to  a  study  of  the  relation  of  the  breed  to 
the  amount  of  pigment  in  the  fat.  This  study  showed  that  the  re- 
lation of  breed  to  milk  fat  coloration  is  a  relative  one,  Holstein  and 
Ayrshire  cows  producing  well-colored  butter  fat  as  well  as  Jerseys 
and  Guernseys  under  proper  feeding  conditions.  It  was  also  shown 
that  under  certain  conditions  there  was  no  difference  in  milk  fat 
coloration  among  the  different  breeds.  These  results  naturally  raised 
the  question  whether  a  similar  study  of  the  body  fat  pigmentation 


4OO  MISSOURI  AGRICULTURAL  EXP.   STA.,  RESEARCH  BULLETIN  NO.   II 


would  lead  to  the  same  conclusions.  It  is  not  uncommon  to  find 
the  body  fat  of  Jersey  and  Guernsey  cows  with  a  high  yellow  color. 
This  has  led  to  a  general  belief  that  this  phenomenon  is  a  character- 
istic of  only  these  breeds  of  cows,  As  a  matter  of  fact  butchers 
and  also  the  consumer  look  with  disfavor  upon  beef  from  these 
animals  on  account  of  this  high  color  of  the  fat.  Although  Table 
3,  above,  shows  very  clearly  that  the  color  of  the  body  fat  of  Jersey 
cows  is  as  much  dependent  upon  the  feed  as  the  color  of  the  milk 
fat,  it  was  nevertheless  important  to  study  the  coloration  of  the 
body  fat,  of  the  different  breeds,  which  had  accumulated  under  ordi- 
nary conditions.  In  this  way  the  normal  breed  relation  could  be 
determined. 

Only  a  few  animals  were  available  for  this  study.  Besides  the 
data  for  the  two  Jersey  cows  given  in  Table  2,  we  have  the  colori- 
metric  study  of  the  body  fat  of  one  Jersey  and  3  Holstein  cows. 
The  data  from  these  animals  is  given  in  Table  4. 

TABLE   4. — RELATION   OF  BREED  TO  COLOR  OF  BODY   PAT. 


Cow  No.  2, 

Cow  No.   207, 

Cow  No.  226, 

Cow  No.  221 

Jersey 

Holstein 

Holstein 

Holstein 

Part  of  body 

Yellow 

Red 

Yellow 

Red 

Yellow 

Red 

Yellow 

Red 

Rib  plate 

54 

1.5 

47 

1.7 

15.0 

1.2 

Crops 

63 

1.8 

14 

1.2 

. 

21.0 

1.2 

Thoracic  cavity 

17 

1.0 

36 

1.5 

'9 

1.0 

6.0 

1.2 

Caul 

50 

1.7 

54 

1.7 

18 

1.1 

12.0 

1.2 

Pelvic  cavity 

47 

1.5 

61 

1.8 

.  . 

10.0 

1.2 

Over  last  rib 

63 

1.8 

17 

1.3 

. 

23.0 

1.2 

Ovaries,  uterus 

62 

1.8 

v 

Chuck 

54 

i'.7 

14 

1.0 

22! 

1.2 

Kidney 

47 

1.5 

64 

1.8 

20. 

1.2 

Stomach 

24 

i!o 

11. 

1.2 

The  most  important  points  presented  in  this  table  are  the  wide 
difference  between  the  color  of  the  fats  of  Holstein  Cow  No.  207 
and  the  other  two  Holstein  cows ;  and  the  wide  difference  between 
the  color  of  the  inside  and  outside  fats  of  Holstein  Cow  No.  207. 
The  first  point  is  possibly  due  to  an  individual  characteristic  of  Cow 
No.  207,  although  it  is  not  known  under  what  conditions  the  fat 
was  formed.  It  should  be  stated  in  connection  with  the  data  of 
Cow  No.  226  that  the  animal  died  in  parturition,  and  the  reason 
so  few  samples  of  fat  are  recorded  is  due  to  the  fact  that  the  animal 
had  no  fat  on  the  body  at  those  particular  places.  In  regard  to  the 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         4OI 

data  on  Cow  No.  207,  it  is  to  be  noticed  that  the  inside  fat  all  had 
a  color  equal  or  greater  than  the  corresponding  fat  of  Jersey  Cow 
No.  2,  while  the  outside  fats  were  uniformly  much  lighter  in  color. 
In  explanation  of  this  result  it  may  be  said  that  milk  cows  are  known 
to  lay  on  fat  first  on  the  inside  of  their  body,  and  we  have  data  to 
show,  not  only  that  this  particular  Holstein  cow  normally  produced 
high-colored  milk  fat  under  favorable  feeding  conditions,  but  also 
that  the  laying  on  of  most  of  the  fat,  whose  color  is  shown  in  the 
table,  was  during  the  summer  when  her  ration  was  largely  fresh 
green  grass. 

This  does  not  hold  true  for  Holstein  Cow  No.  221,  for  much  of 
her  fat  was  also  laid  on  while  on  grass.  It  should  be  added  too 
that  the  milk  fat  of  this  cow  was  never  known  to  have  a  very  high 
color.  This  was  brought  out  especially  in  a  carrot-feeding  experi- 
ment with  this  animal  which  was  reported  in  the  preceding  paper 
of  this  series.  The  maximum  color  obtained  in  that  experiment  was 
practically  the  same  as  the  maximum  color  found  in  her  body  fat. 
There  seems  to  be  a  breed  characteristic  evident  here,  but  owing 
to  the  high  color  readings  obtained  from  Holstein  Cow  No.  207,  it 
may  be  due  to  the  individual  rather  than  to  the  breed. 

Perhaps  the  most  important  point  brought  out  by  this  data  is 
that  the  color  of  the  body  fat  of  any  individual,  regardless  of  breed, 
laid  on  under  given  feeding  conditions  is  practically  the  same  as  the 
color  of  the  milk  fat  under  the  same  conditions. 

Another  point  which  should  be  mentioned  here,  but  which  will 
be  more  readily  understood  in  the  light  of  the  results  which  will 
be  given  in  a  subsequent  paper,  is  that  this  difference  between  indi- 
viduals is  not  due  to  lack  of  carotin  in  the  blood.  The  amount  of 
carotin  in  the  blood  of  Cow  No.  221  at  the  time  of  slaughtering  was 
as  great  as  is  found  in  the  blood  of  a  Jersey  cow  receiving  the  same 
feed. 

Our  data  is  not  sufficiently  extensive  to  warrant  any  conclusions 
as  to  the  normal  difference  in  body  fat  pigmentation  between  the 
different  breeds.  Very  probably  it  is  considerably  greater  than  the 
normal  difference  between  the  milk  fat  pigmentation  of  the  different 
breeds.  The  reason  for  this  is  not  evident  from  our  present  knowl- 
edge of  the  physiology  of  pigmentation.  The  fact  that  such  a  wide 
difference  often  does  exist  is  of  considerable  importance,  however, 
in  explaining  why  the  milk  fat  of  Jersey  and  Guernsey  cows  often 
has  a  higher  color  than  can  be  explained  by  the  character  of  the 
ration.  Reference  was  made  to  this  in  connection  with  the  feeding 
experiments  reported  in  the  preceding  paper  on  the  milk  fat  pigment. 


4O2  MISSOURI  AGRICULTURAL  EXP.   STA.,  RESEARCH  BULLETIN  NO.   II 

It  was  there  shown  that  changing  the  ration  of  a  Jersey  cow  from 
one  rich  in  carotin  and  xanthophylls  to  an  unpalatable  one  very  poor 
in  these  pigments  did  not  result  in  an  immediate  lowering  of  the 
color  of  the  butter  fat,  but  resulted  rather  in  a  gradual  reduction  in 
color,  extending  over  a  considerable  period  of  time.  The  animal 
at  the  same  time  usually  lost  weight.  This  fact  taken  in  connection 
with  the  normal  high  color  of  the  body  fat  of  Jersey  cows  which 
was  brought  out  in  the  present  experiments,  gives  a  clear  explana- 
tion of  the  entire  phenomenon.  The  pigments  of  the  body  fat  were 
being  drawn  upon,  or  rather  the  utilization  of  the  body  fat  for  energy 
liberated  pigments  which  furnished  a  partial  temporary  supply  for 
the  milk  fat.  The  milk  fat  of  cows,  whose  body  fat  lacked  this  high 
color  would,  therefore,  under  similar  conditions  lose  color  very  much 
faster.  The  high  color  of  the  milk  fat  of  Jersey  cows  on  a  nonpig- 
mented  ration  is,  therefore,  due  to  the  fact  that  their  body  fat  has 
a  normal  high  yellow  color. 

THE   PIGMENTS  OF  THE  CORPUS    LUTEUM. 

Viewed  in  the  light  of  the  foregoing  investigations  it  is  not 
surprising  that  Escher  l  has  found  that  the  corpus  luteum  pigment 
belongs  to  the  carotin  group,  thus  establishing  its  identity  with  the 
principal  milk  fat  and  body  fat  pigments.  In  view  of  the  plurality  that 
has  been  established  for  both  the  milk  and  body  fat  pigments  it 
became  at  once  important  to  study  the  corpus  luteum  pigment  in 
this  connection  also.  Only  a  few  corpora  lutea  were  available  for 
the  study,  in  fact  the  ovaries  of  only  six  cows  at  different  times  were 
available  for  examination  and  in  three  cases  only  were  well-devel- 
oped corpora  lutea  found.  Of  Jersey  Cows  No.  21  and  No.  25  slaugh- 
tered at  the  same  time,  only  Cow  No.  25  had  a  corpus  luteum  of  any 
development.  Jersey  Cow  No.  8  and  a  Hereford  cow  were  slaugh- 
tered at  the  same  time  but  only  the  beef  bred  cow  had  a  well-de- 
veloped corpus  luteum.  Cow  No.  207  slaughtered  at  another  time 
had  no  well-developed  corpus  luteum  but  there  were  the  remains 
of  a  number  of  former  corpora  lutea  and  one  just  developing.  Hoi- 
stein  Cow  No.  221  slaughtered  some  time  later,  had  a  well-developed 
corpus  luteum. 

The  investigations  of  the  corpora  lutea  of  the  Jersey  cows,  Nos. 
21  and  25,  were  carried  out  on  the  combined  pigments  previous  to 
the  discovery  of  the  xanthophyll  constituent  of  butter  fat  pigment, 
but  the  data  obtained  is  nevertheless  very  instructive. 

1.    Loc.  cit. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT         403 

The  corpora  lutea  were  carefully  cut  away  from  the  surround- 
ing tissue,  ground  up  with  sand,  and  extracted  with  ether.  In  this 
solvent  and  in  alcohol,  the  pigment  showed  two  absorption  bands 
in  the  blue  part  of  the  spectrum.  Solubility  tests  on  the  alcohol 
solution  showed  that  petroleum  ether  and  carbon  bisulphide  ex- 
tracted almost  all  the  pigment.  That  which  was  not  extracted  was 
treated  with  hot  alcoholic  potash  and  the  soap  extracted  with  ether  in 
which  the  pigment  all  readily  went.  The  saponified  pigment  was  trans- 
ferred to  alcohol  and  freed  from  cholesterol  with  digitonin.  After 
concentrating,  the  cholesterol-free  filtrate  was  extracted  with  carbon 
bisulphide.  Not  all  the  pigment  was  extracted  even  with  two  ex- 
tractions, and  petroleum  ether  extracted  no  color  from  the  remain- 
ing light-yellow  alcoholic  solution.  The  experiments  with  the  sec- 
ondary pigment  were  not  carried  farther  at  this  time  as  the  signifi- 
cance of  its  presence  was  not  appreciated,  but  viewing  the  data  in 
the  light  of  the  results  of  the  milk  fat  and  body  fat  investigations 
it  is  evident  that  a  secondary  xanthophyll  pigment  was  present  here. 
This  has  been  emphasized  because  the  results  of  the  investigations 
subsequently  conducted  were  unfortunately  vitiated  because  of  un- 
expected aldehyde  resin  colorations  which  developed  during  saponi- 
fication.  It  was  shown  by  a  special  study  that  these  reddish  yellow 
bodies  when  present  in  considerable  quantity  are  extracted  from 
the  diluted  alkaline  solutions  by  ether,  but  are  not  readily  extracted 
from  alcohol  by  petroleum  ether.  Consequently  they  interfere  with 
a  proper  study  of  the  pure  pigments.  Such  a  result  was  obtained 
in  the  study  of  the  corpora  lutea  pigments  of  Cow  No.  8  and  the 
Hereford  cow.  The  only  noteworthy  result  of  that  investigation  was 
to  obtain  a  beautiful  rose  colored  unadsorbed  zone  in  a  chromoto- 
gramm  of  a  carbon  bisulphide  solution  of  the  combined  pigment. 
This  solution  showed  three  absorption  bands,  the  measurements  of 
which  are  given  in  Table  5. 

The  next  investigation  was  with  the  corpora  lutea  of  Hoi  stein 
Cow  No.  207.  As  stated  above  there  was  no  well-developed  corpus 
luteum,  the  largest  part  of  the  pigment  obtained  being  from  the  re- 
mains of  several  former  corpora  lutea  which  were  present  as  small 
red  colored  patches  about  the  size  of  a  pin  head.  These  were  care- 
fully cut  out  and  macerated  with  a  little  sand  and  CaSO4  and  extracted 
with  carbon  bisulphide  for  several  hours.  The  solution,  of  about 
25-50  c.  c.  volume,  had  a  deep  orange-red  color,  which  showed"  three 
beautiful  bands,  the  third  band  being  considerably  fainter  than  the 
first  two.  The  measurements  are  given  in  Table  5. 


404  MISSOURI  AGRICULTURAL  EXP.  STAV  RESEARCH  BULLETIN  NO.   II 

A  chromotogramm  of  this  solution  showed  only  one  rose  col- 
ored zone  which  passed  rapidly  through  the  CaCO3  column.  A  little 
of  the  solution  was  evaporated  into  absolute  alcohol  and  after  mak- 
ing the  alcohol  eighty  per  cent,  the  pigment  was  studied  in  regard 
to  its  solubility  properties  toward  petroleum  ether  (b.  p.  30-50°  C) 
and  carbon  bisulphide  respectively.  In  both  cases  the  alcohol  was  left 
absolutely  colorless.  In  this  case  then,  where  the  pigment  was  chiefly 
from  the  remains  of  former  corpora  lutea,  carotin  was  the  only  pig- 
ment present. 

TABLE  5. — ABSORPTION  BANDS  OF  CORPUS  LUTEUM  CAROTIN  IN  CARBON 

BISULPHIDE. 


Bands 

From  Hereford  Cow  No.  8. 
Good  corpus  luteum. 

From  Holstein  Cow  No.  207. 
Remains  of  corpus  luteum.. 

Band  I 
Band  II 
Band  III 

225—242 
262—282 
305—320 

225—242 
262—285 
305—320 

The  final  investigation  was  with  the  well-developed  corpus  lu- 
teum from  Holstein  Cow  No.  221.  Both  ovaries  of  the  cow  were 
ground  up  with  sand  and  plaster  of  paris  and  the  mass  extracted 
with  petroleum  ether  (b.  p.  30-50°  C.)  until  the  extract  was  color- 
less. The  pigment  thus  extracted  was  carefully  differentiated  be- 
tween the  petroleum  ether  and  85  per  cent  alcohol.  No  pigment 
whatever  was  extracted  by  the  alcohol.  The  pigment  was  then  sub- 
mitted to  saponification  with  KOH  after  transferring  to  alcohol.  No 
aldehyde  resin  pigments  formed  during  saponification.  The  pig- 
ment was  extracted  from  the  soap  with  ether.  The  ether  extract 
was  thoroughly  washed  with  water  as  usual  and  then  concentrated  at 
a  low  temperature  with  the  constant  addition  of  petroleum  ether  so 
that  the  pigment  finally  remained  in  petroleum  ether  solution.  This 
solution  was  then  shaken  with  85  per  cent  alcohol.  The  alcohol 
extracted  no  pigment  whatever.  In  this  case  then,  although  a  nor- 
mal corpus  luteum  was  used,  and  the  entire  pigmented  extract  sub- 
mitted to  saponification,  carotin  was  the  only  pigment  present. 

The  result  of  this  study  was  to  confirm  the  results  of  Escher  * 
that  the  corpus  luteum  pigment  is  identical  in  properties  with  caro- 
tin. In  addition  we  have  shown  that  this  pigment,  like  the  prin- 
cipal pigments  of  milk  fat  and  body  fat,  may  have  associated  with 

1.    Loc.  cit. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT         405 

it  small  quantities  of  xanthophyll  pigment..  It  is  possible,  however, 
that  these  xanthophylls  are  present  in  the  fat  which  may  be  extracted 
along  with  the  carotin  of  the  corpus  luteum. 

THE    PIGMENTS    OF   THE    WAXY   SECRETIONS    IN    THE    EARS    AND 
ON  THE  SKIN  OF  JERSEY  COWS. 

It  was  stated  in  the  introduction  that  the  secretions  of  the  skin 
of  Jersey  and  Guernsey  cows  is  often  considered  as  indicating  the 
ability  of  these  breeds  to  secrete  yellow  milk  fat.  It  was  accordingly 
thought  that  a  brief  investigation  of  this  pigment  would  be  of  inter- 
est and  possibly  of  some  scientific  value. 

The  yellow  skin  secretion  of  Jersey  cows  is  especially  abundant 
in  the  ears.  A  few  grams  of  the  yellow  waxy  matter  was  accord- 
ingly scraped  from  the  ears  of  several  pure  bred  Jersey  cows  and 
the  wax  macerated  with  ether,  which  readily  dissolved  away  the 
pigment  and  some  fatty  matter,  giving  a  bright  yellow  solution.  The 
ether  solution  was  concentrated  to  low  volume  and  diluted  with 
about  loo  c.  c.  of  2  per  cent  alcoholic  potash  and  the  solution  boiled 
on  the  steam  bath  for  30  minutes.  The  pigment  was  extracted  from 
the  soap  solution  with  ether  in  the  usual  way.  The  extraction  of 
the  pigment  was  easy  and  complete.  The  ether  solution  was  freed 
from  alkali  as  usual  and  then  diluted  with  some  petroleum  ether.  The 
slightly  cloudy  solution  which  resulted  was  washed  with  water  until 
clear  and  evaporated  into  absolute  alcohol.  The  alcohol  was  now 
diluted  with  petroleum  ether  (b.  p.  30  to  50°  C)  and  water  added 
sufficient  to  cause  separation.  Several  extractions  with  petroleum 
ether  resulted  in  the  division  of  the  original  pigment  into  a  major 
petroleum  ether  soluble  pigment  and  a  minor  pigment  which  could 
not  be  extracted  from  80-90  per  cent  alcohol  with  petroleum  ether. 

The  petroleum  ether  pigment  gave  a  red  orange  carbon  bisulphide 
solution  showing  the  carotin  absorption  bands: 

I.  224—243 

II.  263—287 

III.  303—320 

and  a  beautiful  rose  colored  unadsorbed  zone  in  the  CaCO3  chromoto- 
gramm. 

The  80  per  cent  alcohol  soluble  pigment  which  amounted  tc  two 
or  three  per  cent  of  the  entire  pigment,  gave  a  yellow-orange  car- 
bon bisulphide  solution.  The  solution  showed  only  one  absorption 
band  however,  the  other  bands  being  obscure. 

I.     232—254 


406  MISSOURI  AGRICULTURAL  EXP.  STA.,  RESEARCH  BULLETIN  NO.  II 

It  is  thus  seen  that  the  yellow  pigment  of  the  skin  secretions 
of  the  Jersey  cow  is  identical  with  the  other  yellow  lipochromes  of 
the  body  and  like  them  belongs  chiefly  to  the  carotin  group  of  pig- 
ments. 

THE  BODY  FAT  AND  BLOOD  SERUM  PIGMENTS  OF  THE  NEW-BORN 

CALF. 

Carotin  and  xanthophylls  having  been  found  to  be  normal  con- 
stituents of  the  body  fat  of  cows  which  had  been  fed  green  feeds  or 
other  feeds  containing  an  abundant  amount  of  these  pigments,  an  in- 
teresting question  was  raised  as  to  whether  these  pigments  are  present 
in  the  body  of  the  new-born  calf.  If  these  pigments  should  be  found 
to  be  entirely  absent  from  the  new-born  calf,  additional  proof  would 
therefore  be  offered  that  these  pigments  were  the  result  of  subse- 
quent feeding.  The  presence  of  carotin  and  xanthophylls  in  the  new- 
born calf,  however,  would  not  be  proof  that  these  pigments  cannot 
arise  from  the  food,  but  would  merely  indicate  that  they  were  able 
to  traverse  the  placental  barrier  from  the  mother  whose  body  is 
normally  rich  in  these  pigments.  In  this  connection  the  question 
would  be  especially  interesting  in  view  of  the  fact  that  Mendel  and 
Daniels  l  have  recently  found  that  fat  soluble  dyes,  such  as  Sudan 
III,  do  not  traverse  the  placental  barrier  of  small  animals  such  as 
cats  and  rats,  whose  milk  fat  and  body  fat,  however,  is  readily  tinted 
as  the  result  of  feeding  the  dyes. 

In  order  to  study  this  question,  the  following  experiment  was 
carried  out: 

A  new-born  pure  bred  Jersey  calf  weighing  50  pounds  was  not 
allowed  to  suckle  its  mother  but  was  slaughtered  a  few  hours  after 
its  birth. 

Five  hundred  c.  c.  of  the  blood  was  caught  in  a  cylinder  and 
allowed  to  clot.  After  standing  48  hours,  250  c.  c.  of  serum  was 
obtained.  The  proteins  were  precipitated  from  the  serum  with  al- 
cohol and  were  filtered  off  on  a  Biicher  funnel  with  suction.  They 
had  a  reddish  gray  color.  They  were  rubbed  up  to  a  paste  with 
absolute  alcohol  in  a  mortar  and  then  extracted  with  boiling  abso- 
lute alcohol.  The  extract  was  absolutely  colorless.  The  alcoholic 
filtrate  from  the  precipitated  proteins  had  a  greenish-yellow  color. 
It  was  concentrated  to  50  c.  c.  and  absolute  alcohol  added,  precipitat- 
ing a  little  protein.  The  filtrate  had  a  beautiful  greenish-yellow  color 
but  the  pigment  was  not  extracted  by  carbon  bisulphide,  petroleum 

1.     Jour.  Biol.  Chem.  13  No.  1,  p.  72  (1912).      ~*** 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK   FAT         407 

ether,  or  ether,  but  seemed  to  be  partly  thrown  down  by  lead  acetate 
and  by  saturation  of  a  dilute  alcoholic  solution  with  (NH4)2SO4. 
Acid  mercuric  nitrate  solution  decolorized  the  alcoholic  solution  at 
the  same  time  throwing  down  a  white  precipitate. 

The  only  conclusion  that  can  be  drawn  from  this  experiment 
is  that  the  blood  serum  of  the  new-born  calf  is  free  from  carotin. 
A  small  amount  of  an  unknown  pigment  was  present  which  was 
readily  soluble  only  in  alcohol,  and  insoluble  in  water,  ether,  carbon 
bisulphide,  and  petroleum  ether. 

There  was  practically  no  fat  on  the  body  except  a  little  around 
the  kidneys  and  in  the  tissue  of  the  caul  apron.  In  the  body  the 
latter  tissue  had  a  slight  brownish  color.  All  the  fat  tissue  that 
could  be  obtained  was  ground  up,  rendered  and  filtered.  About  40 
grams  in  all  was  obtained.  The  rendered  fat  had  a  slight  yellow 
color  giving  a  tintometer  reading  in  i-inch  layer  of  4  yellow  and 
.3  red.  When  solid  the  fat  had  a  greenish -yellow  tint.  Thirty  grams 
of  the  fat  was  saponified  with  alcoholic  potash  and  the  soap  extracted 
with  ether.  It  was  possible  to  differentiate  the  small  amount  of 
pigment  thus  obtained  so  that  it  was  about  equally  divided  between 
petroleum  ether  and  80  per  cent  alcohol.  In  carbon  bisulphide  solu- 
tion these  portions  showed  their  relation  to  carotin  and  xanthophyll 
both  in  the  spectroscope  and  chromotogramm.  Both  portions  showed 
two  beautiful  bands  which  measured  as  follows: 

Body  Fat  Carotin  Body  Fat  Xanthophyll 

(In  CS2)  (In  CS2) 

Band     I    225 — 244  Band     I    235 — 250 

Band  II     263—280  Band  II    270—285 

The  results  of  this  experiment  show  that  a  small  amount  of 
carotin  and  xanthophyll  are  present  in  the  body  fat  of  the  new-born 
Jersey  calf.  The  results  present  the  apparent  anomaly,  however,  of 
the  presence  of  the  pigments  in  the  body  fat  and  their  absence  in 
the  blood.  In  explanation  of  this  it  may  be  said  that  the  body  fat 
of  the  new-born  calf,  the  amount  of  which  is  very  small  indeed, 
probably  arises  from  the  fat  of  the  mother,  being  transferred  to  the 
foetus  a  very  small  quantity  at  a  time.  The  small  quantity  which 
would  be  present  in  the  blood  stream  under  these  conditions,  i.  e., 
dissolved  in  fat,  would  not  have  been  detectable  by  the  method  used 
for  the  investigation  of  the  pigments  of  the  blood  serum.  It  is  abso- 
lutely certain  that  there  were  none  of  these  pigments  present  in 
the  blood  serum  in  the  way  in  which  they  are  normally  found  in  the 
mature  animal. 


408  MISSOURI  AGRICULTURAL  EXP.   STAV  RESEARCH  BULLETIN  NO.   II 

The  results  of  the  investigation  are  of  further  value  in  indicat- 
ing that  under  proper  feeding  conditions,  it  might  be  possible  to 
raise  even  a  Jersey  cow  with  practically  none  of  the  characteristic 
carotin  and  xanthophyll  pigments  in  her  body. 

DISCUSSION  OF  RESULTS. 

The  results  of  the  experiments  reported  in  this  paper  are  in 
perfect  accord  with  those  of  the  preceding  paper.  The  discovery  of 
the  carotin  and  xanthophyll  nature  of  the  milk  fat  pigment  would 
lead  quite  naturally  to  the  supposition  that  the  other  lipochrome  pig- 
ments of  the  body  of  the  cow  are  of  the  same  character.  This  sup- 
position was  fully  borne  out  by  the  result  of  experimental  study. 
The  yellow  lipochromes  of  the  body  fat,  of  the  corpus  luteum  and 
of  the  skin  secretions  were  found  to  be  composed  principally  of  car- 
otin with  one  or  more  minor  xanthophyll  constituents. 

In  addition  to  the  establishment  of  the  chemical  relation  of  these 
pigments  to  the  carotin  and  xanthophyl  of  green  plants  in  the  case 
of  the  body  fat  at  least  it  has  been  possible  to  show  that  the  pig- 
ments are  derived  from  the  food  in  a  manner  identical  with  pig- 
ments of  the  milk  fat.  The  carotin  and  xanthophylls  of  the  corpus 
luteum  and  skin  secretions  must  therefore  be  derived  from  the  same 
source. 

Viewing  the  results  from  a  physiological  standpoint,  it  is  seen 
that  the  establishment  of  such  a  source  for  these  pigments  and  the 
ease  with  which  they  are  therefore  increased  and  decreased,1  throws 
great  doubt  upon  any  physiological  significance  which  these  pigments 
have  been  supposed  to  exert  in  the  animal  body.  In  the  case  of  the 
corpus  luteum  for  instance,  the  accumulation  of  the  carotin  during 
the  formation  of  this  body  is  merely  a  phenomenon  incidental  to  the 
rupture  of  the  Graafian  follicle  and  the  subsequent  formation  of  the 
cellular  tissue  around  the  central  blood  clot,  and  to  the  fact  that  the 
blood  serum  is  normally  very  rich  in  carotin,  as  will  be  shown  in 
the  following  paper.  This,  of  course,  does  not  explain  the  mechanism 
of  the  accumulation  of  the  carotin-containing  cells  around  the  ruptured 
Graafian  follicle.  The  chemical  combination  of  the  carotin  in  the 
blood  serum  is  no  doubt  of  importance  in  this  connection. 

The  popular  opinion  that  the  body  fat  of  Jersey  cows  is  nor- 
mally characterized  by  a  higher  yellow  color  than  Holstein  cows  has 
been  at  le'ast  partially  confirmed  by  experimental  study,  although  it 

1.  This  is  especially  true  of  the  milk  fat  and,  as  will  be  shown  in  the 
succeeding  paper,  the  blood  serum. 


CAROTIN,  THE  PRINCIPAL  YELLOW   PIGMENT  OF   MILK   FAT         409 

was  found  that  Holstein  cows  may  also  possess  high-colored  body 
fat.  At  least  there  seems  to  be  more  breed  characteristic  in  this 
respect,  than  in  the  case  of  the  pigmentation  of  the  milk  fat.  There 
is  no  foundation,  however,  for  the  belief  that  beef  has  a  lower  value 
because  its  fat  has  a  high  color.  If  this  pigment  is  the  same  as  is 
demanded  by  the  consumer  for  butter,  why  should  not  beef  with  high- 
colored  fat  also  be  more  desirable  ?  It  is  recognized  of  course  that 
some  of  the  unfavorable  attitude  toward  beef  with  highly  colored 
fat  arises  partially  from  the  fact  that  it  indicates  that  the  beef  prob- 
ably came  from  a  dairy  cow.  The  two  ideas  are  nevertheless  very 
closely  associated. 

The  normally  high  color  of  the  body  fat  of  Jersey  cows  and 
also  of  those  of  the  Guernsey  breed,  explains  why  cows  of  these  breeds 
often  appear  to  be  producing  well-colored  butter  on  a  ration  deficient 
in  carotin  and  xanthophylls.  Several  statements  in  regard  to  this 
have  already  been  made.  This  will  bear  repetition,  however,  because 
the  subject  is  an  important  one.  Briefly,  it  may  be  said  that  when 
cows  whose  body  fat  has  a  high  yellow  color  are  put  upon  a  ration 
deficient  in  carotin  and  xanthophylls  and  also,  as  is  usually  the  case 
with  such  rations,  deficient  in  food  value,  the  body  fat  is  called  upon 
to  furnish  energy  value  for  the  animal  and  also  in  many  cases  to 
supplement  the  food  digestion  products  in  the  production  of  milk 
fat.  It  is  readily  seen  that  in  such  cases  an  important  source  is 
opened  up  for  pigments  for  the  milk  fat.  Just  how  important  this 
source  could  be  would  depend  upon  the  amount  of  highly  colored 
body  fat  available  for  the  needs  of  the  body,  and  upon  the  rapidity 
with  which  it  would  be  used  up.  If  our  experimental  data  are  cor- 
rect showing  that  the  inside  fats,  such  as  the  caul  fat  and  rib  plate 
fat,  are  the  first  drawn  upon  in  starvation  of  this  class  of  animals, 
then  the  amount  of  available  highly  colored  fat  would  be  rather 
large.  Dairy  cows  usually  have  a  fairly  abundant  amount  of  these 
fats,  especially  the  caul  fat.  It  is  thus  readily  seen  that  a  continuous 
drawing  upon  these  inside  fats  for  a  long  period  of  time  would  result 
in  a  very  slow  and  gradual  reduction  of  the  color  of  the  milk  fat. 
The  deduction  that  the  animal  was  actually  producing  colored  milk 
fat  on  a  carotin-xanthophyll-free  ration  would,  therefore,  be  quite  nat- 
ural but  nevertheless  entirely  false. 

In  a  similar  manner  it  is  readily  seen  why  the  breeders  of  Jer- 
sey and  Guernsey  cattle  have  been  led  to  believe  that  the  yellow  skin 
secretions  of  these  breeds  are  indicative  of  their  ability  to  produce 
yellow  milk  fat.  It  is  interesting  to  find  that  the  yellow  pigments 
of  these  secretions  are  carotin  and  xanthophylls.  It  should  be  clearly 


4IO  MISSOURI  AGRICULTURAL  EXP.  STA.,  RESEARCH  BULLETIN  NO.  II 

borne  in  mind,  however,  that  the  only  indication  that  a  cow  will  secrete 
yellow  milk  fat  is  that  the  food  contains  an  abundance  of  carotin  and 
xanthophylls. 


SUMMARY. 

1.  The  yellow  lipochrome  of  the  body  fat,  corpus  luteum  and 
skin  secretions  of  the  cow,  like  the  lipochrome  of  butter  fat,  is  com- 
posed principally  of  a  pigment  whose  physical  and  chemical  properties 
are  identical  with  the  carotin  of  green  plants.    The  same  pigment  may 
have  associated  with  it  one  or  more  minor  constituents  whose  physi- 
cal and  chemical  properties   are  identical  with  the  xanthophylls  of 
green  plants. 

2.  The  carotin  and  xanthophyll  pigments  of  the  body  fat  are 
derived  from  the  food  of  the  cow.     The  body  fat  of  Jersey  cows 
formed  on  a  ration  deficient  in  carotin  and  xanthophylls,  is  devoid 
of  color. 

3.  The  body  fat  of  Jersey  and  Guernsey  cows  is  usually  char- 
acterized by  a  higher  yellow  color  than  cows  of  other  breeds.     This 
is  of  great  importance  in  explaining  why  cows  of  these  breeds  may 
sometimes   show   a  much   slower  elimination  of  the  pigment   from 
milk  fat  on  a  non-pigmented  ration,  as  during  the  winter  months. 
In  these  cases  the  body  fat  furnishes  a  supplementary  source  of  pig- 
ments for  the  milk  fat. 

4.  The  yellow  body  fat  of  Jersey  and  Guernsey  cows  should 
not  be  a  point  against  the  use  of  these  animals  for  beef.     The  pig- 
ments here  are  the  same  as  those  for  which  the  consumer  will  pay  a 
higher  price  when  present  in  butter. 

5.  The  breeders  of  Jersey  and   Guernsey  cattle  are  probably 
correct  in  their  belief  that  the  yellow  skin  and  skin  secretions  of 
these  animals  are  characteristic  of  the  breeds.    It  is  not  correct,  how- 
ever, that  this  characteristic  is  indicative  of  the  ability  of  these  ani- 
mals to  secrete  yellow  milk  fat  under  all  conditions.    The  only  indi- 
cation of  this  is  whether  the  food  contains  an  abundance  of  carotin 
and  xanthophylls. 

6.  The  blood  serum  of  the  new-born  Jersey  calf  is  free  from 
carotin  and  xanthophylls.     The  small  amount  of  fat  on  the  body  is 
tinted  very  faintly  with  these  pigments. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT        411 

BIBLIOGRAPHY. 

1.  Escher:    Zeit.  f.  Physiol.  Chem.  83,  p.  198  (1913). 

2.  Mendel  and  Daniels:    Jour.  Biol.  Chem.  13,  No.  i,  p.  72  (1912). 
Salmon"  1898,  Article  XV,  p.  159. 

3.  Newbigin:  D.  Noel  Paton,  "Report  of  Inves.  on  Life  History  of 

4.  Tswett:    Ber.  d.  Deut.  Botan.  Gessel.  24,  pp.  316,  384   (1906). 

5.  Tswett:  Ber.  d.  Deut.  Botan.  Gessel.  29,  p.  630  (1911). 

6.  Willstatter  &  Escher:     Zeit.  f.  PhysioL  Chem,  76,  pp.  214-225 
(1912). 

7.  Willstatter  and  Mieg:   Ann.  d.  Chem.  355,  p.  i   (1907). 


CAROTIN— THE  PRINCIPAL  NATURAL  YELLOW  PIGMENT 
OF  MILK  FAT— Part  IV,  * 


A.  The  Yellow  Pigment  of  Blood  Serum. 

B.  Carotin  and  Xanthophylls  During  Digestion. 

C.  The  Pigments  of  Human  Milk  Fat. 

LEROY  S.  PALMER  and  C.  H.  ECKLES. 

A.     THE  YELLOW  PIGMENT  OF  BLOOD  SERUM 

Very  few  investigations  have  dealt  with  the  so-called  lutein  of  the 
blood  serum.  Thudichum  1  was  the  first  to  mention  it  and  classify 
it  as  a  lutein.  Schunck,2  a  number  of  years  later,  showed  that  the 
lutein  of  fowl  serum  was  spectroscopically  identical  with  the  L. 
xanthophyll  which  he  isolated  from  yellow  flowers  and  green  plants. 
Halliburton  3  also  studied  the  lutein  of  the  serum  of  the  hen,  but  the 
pigment  isolated  by  him  had  evidently  lost  its  spectroscopic  properties 
in  view  of  Schunck's  investigation.  Finally,  Krukenberg4  extracted 
the  lutein  from  ox  serum  by  shaking  with  amyl  alcohol.  The  extract 
showed  two  absorption  bands.  He  used  the  designation  lipochrome 
for  the  pigment.  His  work  is  usually  mentioned  in  the  present  text 
books  of  physiological  chemistry. 

Recent  investigations  in  connection  with  various  animal  luteins 
or  lipochromes  have  shown  that  they  may  be  classified  as  belonging 
to  the  widely  distributed  carotin  or  xanthophyll  groups  of  pigments 
of  the  vegetable  world.  Willstatter  and  Escher5  have  identified  the 
pigment  of  egg  yolk  as  an  isomer  of  the  crystalline  xanthophyll  of 
green  plants ;  and  Escher  6  has  shown  that  the  principal  corpus  luteuni 
pigment  is  identical  with  the  carotin  of  plants.  Extending  this  work 

*See  Research  Bulletin  No.  9,  p.  312,  for  statement  of  Co-operation  with 
TJ.  S.  Dept.  of  Agriculture. 

1.  Proc.  Roy.  Soc.  17,  p.  253   (1869). 

2.  Proc.  Roy.  Soc.  72,  p.  165   (1903). 

3.  Jour.  Physiol.  7,  p.  324    (1886). 

4.  Sitz,  Ber.  d.  Jen.  Gessel.    (1885). 

5.  Zeit.  f.  Physiol.  Chem.  76,  pp.  214-225  (1912). 

6.  Zeit  f.  Physiol.  Chem.  83,  p.  198  (1913). 

(415) 


4l6       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

we  have  shown  that  the  yellow  lipochromes  of  the  milk  fat  and  body 
fat  of  cows  are  also  composed  principally  of  carotin,  altho  both  have 
associated  with  them  one  or  more  minor  xanthophyll  constituents.  In 
addition  we  have  shown  conclusively  that  these  pigments  originate 
from  the  food  of  the  cow.  They  are  therefore  not  products  of  animal 
synthesis  but  merely  substances  assimilated  with  the  digestion  products 
of  the  food  and  subsequently  secreted  in  the  milk  fat  or  laid  up  in  the 
body  fat.  The  studies  leading  to  these  results  are  given  in  the  two 
preceding  papers  of  this  series.1 

Up  to  this  time  the  experimental  evidence  pointing  to  the  above 
stated  physiological  relation  between  the  carotin  and  xanthophylls  of 
plants  and  the  pigments  of  butter  fat  and  body  fat  has  been  based  upon 
feeding  experiments  in  which  the  relation  between  the  amount  of  these 
pigments  in  the  food  and  the  color  of  the  milk  fat  and  body  fat  was 
carefully  studied.  It  was  recognized  however  that  the  evidence  would 
not  be  absolutely  complete  until  the  means  of  transporting  the  food 
pigments  to  the  milk  fat  and  body  fat  could  be  established.  A 
close  study  of  the  yellow  lipochrome  of  the  blood  serum,  in  a  man- 
ner similar  to  the  preceding  studies  of  the  milk  fat  and  body  fat 
pigments,  naturally  seemed  to  offer  the  most  ready  means  of  establish- 
ing the  physiological  relation  between  the  plant  pigments  and  the  lipo- 
chromes of  milk  fat  and  body  fat. 

The  present  investigation  was  therefore  undertaken  for  the  pur- 
pose of  studying  the  yellow  lipochrome  of  the  blood  serum  in 
regard  to  its  chemical  and  physiological  relations  to  the  carotin  and 
xanthophylls  of  green  plants  and  to  these  pigments  when  found  in  the 
milk  fat  and  body  fat  of  the  cow.  It  was  believed  that  this  investigation 
would  serve  the  twofold  purpose  of  establishing  the  connecting  physi- 
ological link  between  these  plant  and  animal  pigments  and  also  scien- 
tifically classifying  the  blood  serum  lutein  of  the  cow  which  pigment 
has  never  been  the  subject  of  close  investigation. 

METHODS  OF   IDENTIFICATION. 

The  methods  used  for  identifying  the  pigment  of  the  blood 
serurn  were  the  same  as  were  used  in  the  study  of  the  milk  fat  and 
body  fat  pigments.  They  consisted  in  the  application  to  the  isolated 
pigment  of  the  characteristic  physical  and  chemical  properties  of 
carotin  and  xanthophylls.  These  properties  were  the  position  of  the 
spectroscopic  absorption  bands,  the  relative  solubility  toward  petroleum 
ether  and  80  to  90  per  cent  alcohol,  and  the  adsorption  affinity  toward 

1.     Also  Jour.  Biol.  Chem.  17,  No.  2,  pp.  191,  211  (1914). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT 


417 


calcium  carbonate.  A  detailed  description  of  these  properties  when 
applied  to  both  the  plant  carotin  and  xanthophylls  and  the  pigments 
of  milk  fat,  body  fat,  corpus  luteum  and  skin  secretions  of  the  cow 
was  given  in  the  two  preceding  bulletins  1  of  this  series,  and  need  not 
be  repeated  here.  The  measurements  of  the  spectroscopic  absorption 
bands  of  the  carotin  and  xanthophylls  which  are  used  for  comparison 
were  made  according  to  an  arbitrarily  fixed  and  standard  scale.  It 
may  not  be  out  of  place  therefore,  to  repeat  here  a  table  which  was 
given  in  the  paper  immediately  preceding  this  one,  showing  these 
standard  measurements.  This  table  is  given  below  as  Table  I.  The 
measurements  in  carbon  bisulphide  solution  only  are  given. 

TABLE  No.  1. — SPECTROSCOPIC  STANDARDS  OF  CAROTIN  AND  XANTHOPHYLLS. 


Pigment 

Solvent 

Measurements  of  absorption  bands 

Band  I 

Band  II 

Band  III 

Carotin 
Xanthophyll 

CS2 
CS2 

225—242! 
233—253: 

261—278 
272—291 

301—319 
312—330 

METHODS  OF  ISOLATION. 

The  study  of  the  blood  serum  lutein  required  considerable  pre- 
liminary study  of  methods  of  isolation.  The  amyl  alcohol  method  of 
Krukenberg  2  was  not  considered  suitable  on  account  of  the  high  boil- 
ing point  of  the  solvent.  The  method  used  by  Schunck  3  seemed  to  be 
much  better  suited  for  the  work.  He  precipitated  the  proteins  from 
the  serum  with  alcohol,  and  as  the  proteins  carried  down  the  lutein  he 
was  able  to  isolate  it  by  extracting  the  precipitated  proteins  with  boil- 
ing absolute  alcohol.  Preliminary  investigations  of  the  blood  serum 
lutein  using  Schunck's  method  showed,  however,  that  it  was  applicable 
only  to  serum  free  from  dissolved  red  blood  corpuscles.  When 
hemoglobin  was  present  it  was  always  carried  down  with  the  protein 
and  some  of  the  red  color  dissolved  in  the  subsequent  alcohol  extract. 
In  addition,  the  method  did  not  seem  to  be  a  quantitative  one,  some  of 
the  lutein  invariably  being  found  in  the  dilute  alcoholic  filtrate  from 
the  precipitated  proteins.  These  investigations  showed  however,  that 
in  every  case  both  petroleum  ether  and  carbon  bisulphide  almost  quanti- 

1.  Research  Bulletins  Nos.  10  and  11,  Missouri  Agr.  Exp.  Sta.;  also  Jour. 
Biol.  Chem.    17,  pp.   191,   211    (1914). 

2.  Loc.  cit. 

3.  Loc.  cit 


4l8       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

tatively  extracted  the  yellow  pigment  from  its  alcoholic  solution  on 
dilution  with  a  little  water.  This  result  at  once  indicated  the  carotin 
nature  of  the  blood  serum  lutein,  and  this  was  confirmed  by  the  experi- 
ments reported  below. 

The  methods  used  for  isolating  the  pigment  in  these  studies  varied 
somewhat  in  detail  but  were  all  based  upon  a  preliminary,  more  or 
less  complete  dessication  of  the  blood  serum  by  calcium  sulphate 
(plaster  of  Paris).  The  details  are  given  in  connection  with  the  report 
of  the  experiments. 

CHEMICAL    IDENTIFICATION    OF   THE    PIGMENT. 

The  following  experiments  were  conducted  to  show  the  chemical 
relation  of  the  blood  serum  lutein  to  the  carotin  and  xanthophylls. 

Experiment  I. 

About  20  cubic  centimeters  of  golden  yellow  serum  from  Jersey 
Cow  No.  8*  was  mixed  with  plaster  of  Paris  until  almost  dry,  dried 
for  a  few  minutes  on  the  steam  bath,  the  mass  pulverized,  and  shaken 
with  successive  volumes  of  petroleum  either  in  an  Erlenmeyer  flask 
until  no  more  color  appeared  in  the  petroleum  ether.  The  extract  was 
light  yellow  in  color  and  no  color  was  extracted  from  the  concentrated 
solution  by  80  per  cent  alcohol.  The  plaster  of  Paris  mass  was  now 
shaken  with  successive  proportions  of  petroleum  ether  containing  10  per 
cent  absolute  alcohol,  until  the  extraction  was  colorless.  The  resulting 
extract  had  a  deep  yellow  color  containing  many  times  as  much  pigment 
as  the  extract  with  petroleum  ether  alone.  This  solution,  after  con- 
centration, was  extracted  with  80  per  cent  alcohol.  Apparently  no 
color  was  extracted.  The  petroleum  ether  solutions  were  combined, 
evaporated  to  dryness  and  the  residue  dissolved  at  once  in  carbon 
bisulphide  giving  a  deep  red-orange  solution  which  showed  3  absorp- 
tion bands,  Band  III  being  much  fainter  than  the  other  two.  The 
measurements  of  the  bands  are  given  in  Table  No.  2. 

Experiment  II. 

Fifty  cubic  centimeters  of  the  same  serum  was  completely  dessi- 
cated  with  plaster  of  Paris  and  the  pulverized  mass  shaken  with  absolute 
alcohol  and  ether  until  no  more  color  was  extracted.  The  ether  was 
distilled  off  and  the  golden-yellow  alcoholic  solution  saponified  with 

*Note: — The  serum  in  this  case  and  in  all  subsequent  cases  was  obtained 
by  allowing  the  freshly  drawn  blood  to  clot  in  a  tall  cylinder  or  jar  and 
the  serum  which  pressed  out  on  standing  syphoned  off  into  glass  stoppered 
bottles. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  419 

KOH.  After  dilution  with  water,  the  soap  was  extracted  with  ether. 
The  ether  was  washed,  filtered  and  evaporated  into  absolute  alcohol. 
The  alcohol  was  diluted  to  an  80  per  cent  solution  and  extracted  with 
petroleum  ether  (b.p.3O-5o°C.)  until  no  more  color  was  extracted.  The 
alcohol  layer  was  left  with  quite  a  little  color,  but  by  far  the  greatest 
part  of  the  color  was  in  the  petroleum  ether  extracts.  The  petroleum 
ether  soluble  pigment  gave  a  deep  red  carbon  bisulphide  solution  which 
showed  3  absorption  bands,  the  third  being  faint.  The  measurements 
of  the  band  are  given  in  Table  No.  2. 

The  80  per  cent  alcohol  soluble  pigment  showed  no  clear  absorp- 
tion bands. 

Experiment  III. 

Fifty  cubic  centimeters  of  the  same  serum  was  mixed  to  a  thick 
paste  with  plaster  of  Paris,  and  the  pasty  mass  shaken  thoroughly 
with  700  c.c.  of  hot  95  per  cent  alcohol.  All  the  color  was  extracted, 
a  second  extraction  with  fresh  alcohol  being  colorless.  The  yellow 
extract  was  concentrated  to  100  c.c.  An  equal  volume  of  10  per  cent 
alcoholic  potash  was  added  and  the  solution  boiled  on  the  steam  bath 
for  one  hour.  No  aldehyde  resin  pigments  formed.  The  alkaline 
solution  was  diluted  with  3  volumes  of  distilled  water  and  extracted 
with  two-thirds  of  its  volume  of  ether.  All  the  color  was  extracted 
by  the  one  extraction.  After  washing  and  filtering,  the  golden-yellow 
extract  was  evaporated  to  dryness  and  the  residue  taken  up  at  once 
with  petroleum  ether  (b.p.3O-5o°C.)  This  solution  was  now  thor- 
oughly shaken  with  80  per  cent  alcohol  until  no  more  color  was  ex- 
tracted. Fresh  petroleum  ether  extracted  a  little  color  from  the  alco- 
holic extract,  which  was  not  re-extracted  by  fresh  80  per  cent  alcohol. 
The  blood  serum  lutein  was  now  divided  into  a  major  and  minor 
pigment,  the  major  being  insoluble  in  80  per  cent  alcohol  in  the  pres- 
ence of  petroleum  ether  and  the  minor  being  insoluble  in  petroleum 
ether  in  the  presence  of  80  per  cent  alcohol. 

The  petroleum  ether  soluble  pigment  had  a  blood-red  color  in  CS2 
solution  and  showed  3  absorption  bands,  the  measurements  of  which 
are  given  in  Table  No.  2. 

Analyzed  chromotographically  it  passed  through  CaCO3  un- 
adsorbed  as  a  beautiful  rose-colored  area,  leaving  no  adsorbed  zones. 

The  80  per  cent  alcohol  soluble  pigment  was  transferred  to  ether 
by  diluting  its  alcoholic-ether  solution  with  much  water,  and  from 
the  ether  to  carbon  bisulphide  after  evaporation  of  the  former.  In 
carbon  bisulphide  it  gave  an  orange-yellow  solution  showing  two 
absorption  bands  in  the  25  m.m.  cell.  The  measurements  of  the  bands 
are  given  in  Table  No.  2. 


42O       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

Chromotographic  analysis  showed  the  presence  of  only  one  pig- 
ment which  very  slowly  passed  through  the  CaCO3  as  a  yellow  zone. 

Experiment  IV. 

Serum  from  Jersey  Cow  No.  2,  to  the  amount  ot  275  cubic 
centimeters,  was  dessicated  with  a  little  more  than  the  calculated 
amount  of  plaster  of  Paris  necessary  to  take  up  the  water,  and  after 
setting  over  night  the  hard  mass  was  pulverized  in  a  mortar.  The 
powder  was  moistened  with  95  per  cent  alcohol  and  shaken  with 
petroleum  ether  (b.p.3O-5o°C.)  until  the  petroleum  ether  extracted 
no  more  color,  and  then  with  ether  until  that  extract  was  colorless.1 
The  petroleum  ether  extract  was  concentrated  to  50  c.c.  and  the 
solution  added  to  the  alcohol-free  ether-alcohol  extract,  which  had 
been  concentrated  to  about  150  c.c.  An  equal  volume  of  4  per  cent 
alcoholic  potash  solution  was  now  added  to  the  combined  ethereal 
solutions,  the  ethers  evaporated  off  and  the  alcoholic  solution  boiled 
on  the  steam  bath  for  a  few  minutes.  The  pigment  was  then  ex- 
tracted from  the  alkaline  alcoholic  solution  in  the  usual  way  and  when 
in  alcoholic  solution  was  analyzed  with  respect  to  petroleum  ether 
and  eighty  to  ninety  per  cent  alcohol.  Two  pigments  were  thus 
obtained  with  proportions  of  perhaps  95  and  5  per  cent  of  the  total. 

The  petroleum  ether  soluble  pigment  gave  a  red  colored  residue 
which  dissolved  instantly  in  carbon  bisulphide  with  a  blood-red  color, 
and  showed  the  most  beautiful  absorption  bands  yet  seen  for  this 
pigment.  Three  bands  were  visible,  the  third  band  being  considerably 
fainter  than  the  other  two  bands  but  clear  and  distinct.  The  measure- 
ments are  given  in  Table  No.  2. 

1.  The  mechanism  of  this  method  of  obtaining  the  blood  serum  pigment 
is  so  interesting,  its  advantages  so  striking  and  its  results  so  satisfactory, 
that  it  is  worthy  of  some  discussion. 

It  appears  that  the  addition  of  just  sufficient  alcohol  (either  absolute  or 
95  per  cent)  to  thoroughly  moisten  the  dessicated  serum  liberates  the  main 
lutein  pigment  in  such  a  way  that  when  the  moistened  mass  is  shaken  with 
petroleum  ether  the  result  is  the  same  as  if  an  80  per  cent  to  90  per  cent 
alcoholic  solution  of  the  isolated  pigment  is  shaken  with  petroleum  ether. 
There  is  the  additional  advantage  however,  that  the  CaSO4  prevents  the 
formation  of  emulsions  and  holds  the  alcoholic  solution  so  firmly  fixed  in  the 
paste  that  the  petroleum  ether  can  be  poured  away  and  the  use  of  a 
separatory  funnel  be  entirely  dispensed  with.  When  all  the  pigment  more 
soluble  in  petroleum  ether  than  in  80  per  cent  alcohol  is  thus  extracted, 
any  pigment  which  remains  can  be  readily  extracted  with  ether  which  mixes 
readily  with  the  dilute  alcohol.  The  pigment  thus  extracted  can  be  readily 
freed  from  alcohol  by  shaking  with  water  leaving  the  last  as  well  as  the 
first  pigment  extracted,  in  low  boiling  point  solvents,  an  additional  decided 
advantage  in  view  of  the  ease  with  which  they  are  oxidized.  It  should  be 
added  however  that  the  method  for  extracting  the  pigment  more  soluble  in 
alcohol  than  in  petroleum  ether  does  not  apply  well  for  serum  containing 
much  haemoglobin  for  in  this  as  well  as  all  other  alcohol  methods  the  red 
pigment  is  somewhat  soluble  in  the  dilute  alcohol. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT 


421 


Analyzed  by  means  of  a  chromotogramm  the  solution  showed 
only  a  wide,  quickly  filtering,  unadsorbed,  rose-colored  zone. 

The  80  per  cent  alcohol  soluble  pigment  was  transferred  to  ether 
and  then  to  carbon  bisulphide,  giving  in  the  latter  an  orange-yellow 
solution  showing  one  band  and  much  end-absorption.  In  the  chromo- 
togramm it  showed  2  zones  close  together,  an  upper  orange  zone  and 
a  lower  canary  yellow  zone.  The  carbon  bisulphide  solution  of  the 
orange  zone  showed  2  absorption  bands  and  end-absorption.  The 
measurements  are  given  in  Table  No.  2. 

TABLE  No.  2. — SPECTEOSCOPIC  ABSORPTION  BANDS  OF  BLOOD  SERUM  CAROTIN  AND 

XANTHOPHYLLS. 


Absorption  bands 

Experiment 

Solvent 

No. 

Carotin 

Xanthophylls 

1 

I. 

225—242 

CS2 

II. 

263—286 

III. 

305—322 

I. 

223—243 

2 

CS2 

II. 

262—285 

III. 

302—325 

I. 

225—242 

I.     232—254 

3 

CS2 

II. 

263—286 

II.     273—295 

III. 

305—325 

III. 

4 

CS2 

I. 

223—242 

I.     232—252 

II. 

262—286 

II.     270—292 

III. 

300—320 

III.     310— 

DISCUSSION  OF  EXPERIMENTS. 

It  must  be  concluded  from  the  above  experiments  that  the  princi- 
pal lipochrome  of  the  blood  serum  of  the  cow  is  identical  with  that 
of  the  milk  fat,  body  fat,  and  corpus  luteum,  and  as  in  the  case  of 
these  pigments,  with  the  carotin  of  green  plants. 

It  appears  also  from  the  above  investigations  that  a  small  portion 
of  the  blood  lutein  pigment  is  composed  of  xanthophylls.  It  was  found 
to  be  much  more  difficult  to  show  their  presence  in  the  blood  serum 
than  in  the  body  fat  or  butter  fat.  The  reason  for  this  is  not  perfectly 
clear  but  a  close  study  of  the  investigations  throws  some  light  on  the 
question.  It  will  be  noticed  that  it  required  complete  extraction  of 


422       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

a  comparatively  large  amount  of  serum  with  ether  and  subsequent 
saponification  of  the  fat  thus  extracted  to  really  demonstrate  the  pres- 
ence of  xanthophylls.  It  is  a  well-known  physiological  fact  that  the 
proportion  of  fat  in  blood  serum  is  comparatively  small.*  When 
coupled  with  the  above  observations,  this  seems  to  indicate  a  relation 
between  the  xanthophylls  and  the  fat  carried  by  the  blood.  Some 
observations  which  will  be  reported  later,  in  connection  with  the 
fate  of  the  carotin  and  xanthophylls  during  digestion,  will  furnish 
more  evidence  in  this  same  direction. 

PHYSIOLOGICAL  RELATION  BETWEEN  CAROTIN  OF  BLOOD  SERUM 

AND  FOOD  OF  COW. 

After  establishing  the  chemical  relation  between  the  principal 
blood  serum  lipochrome  and  the  carotin  of  the  food,  it  became  im- 
portant to  establish  a  similar  relation  from  a  physiological  standpoint. 
Very  fortunately  this  was  recognized  previous  to  conducting  some  of 
the  important  feeding  experiments  which  showed  the  relation  between 
the  color  of  milk  fat  and  the  food  of  the  cow,  and  which  were  re- 
ported in  Research  Bulletin  No.  io/  Missouri  Agr.  Exp.  Sta.,  the 
second  bulletin  in  this  series.  It  was  accordingly  arranged  to  study 
the  variation  in  the  amount  of  carotin  and  xanthophylls  in  the  blood 
serum  during  portions  of  these  feeding  experiments.  In  this  way  it 
could  be  determined  what  relation  exists  between  the  amount  of  carotin 
and  xanthophylls  in  the  blood  serum  and  the  amount  of  these  pigments 
in  the  milk  fat,  as  well  as  the  relation  between  the  amount  of  carotin 
and  xanthophylls  in  the  serum  and  amount  of  these  pigments  in 
the  food.  Such  a  study  required  the  devising  of  some  method  of 
analysis  whereby  the  color  of  the  various  blood  serums  could  be  com- 
pared with  each  other  and  also  with  the  color  of  the  butter  fat. 
The  following  method  was  adopted. 

In  the  case  of  live  cows  whose  blood  was  to  be  tested,  a  trocar 
was  inserted  in  the  jugular  vein  and  200  to  250  c.  cm.  of  blood  drawn 
off  into  a  glass  cylinder.  As  soon  as  the  blood  had  clotted  and  suf- 
ficient serum  had  pressed  out,  two  10  cc.  portions  were  pipetted  off 
and  carefully  dessicated  with  an  excess  of  plaster  of  Paris.  The 
powdered  mass  was  moistened  with  absolute  alcohol,  and  the  color 
extracted  immediately  by  shaking  with  the  selected  solvent  until  color- 
less. For  one  sample  the  solvent  was  ether  and  for  the  other  sample 
the  solvent  was  petroleum  ether.  In  all  the  studies  the  petroleum 

*Nate: — Hammerstein  gives  .1  to  .7%. 

1.    Also  Jour.  Biol.  Chem.  17,  p.  19!  (1914). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT 


423 


ether  extract  proved  to  be  the  easiest  to  handle  as  it  was  practically 
free  from  water.  The  extract  in  each  case  was  carefully  evaporated 
to  a  volume  of  i  to  2  cubic  centimeters  and  then  made  up  to  12^/2 
cubic  centimeters  with  absolute  alcohol,  this  volume  being  just  suf- 
ficient to  fill  the  one-inch  cell  of  the  Lovibond  Tintometer.  The 
solutions  were  analyzed  at  once  in  the  Tintometer  and  their  color 
readings  recorded.  Duplicate  determinations  were  thus  obtained. 
This  was  considered  necessary  since  the  method  was  not  free  from 
error  due  to  possible  bleaching  of  the  extracted  pigments.  The  entire 
procedure  was  carried  out  as  quickly  as  possible.  The  results  of  the 
duplicate  determinations  were  averaged. 

The  first  series  of  observations  of  the  color  of  the  milk  fat  and 
blood  serum  corresponding  to  various  pigmented  rations  was  made 
with  Ayrshire  cow  No.  301.  These  feeding  experiments  and  the  result- 
ing variation  in  the  color  of  the  milk  fat  were  shown  in  detail  in 
Tables  12,  13  and  14  of  the  preceding  bulletin  dealing  with  the  milk 
fat  pigment.  The  relation  between  the  character  of  the  ration,  and 
the  color  of  the  milk  fat  and  blood  serum  at  stated  intervals  during 
the  feeding  experiments  is  shown  in  Table  No.  3,  below. 

TABLE  No.  3. — THE  RELATION  OF  THE  CHARACTER  OF  THE  RATION  TO  THE  COLOR 

OF  THE  MILK  FAT  AND  BLOOD  SERUM. 

AYRSHIRE  Cow  No.  301. 


Date  of 
sample. 

Feed  of  cow 

Butterfat 

Serum 

Yellow 

Red 

Yellow 

Red 

1913 

Jan.  7 
Jan.  24 
Feb.  7 

Mar.  1 
Mar.  6 
Mar.  27 

Cottonseed  meal  and   cotton- 
seed hulls  only 

1.3 
1.2 

2.0 
24.0 

24.0 
7.0 

0.4 
0.4 

0.5 
1.3 

1.4 
1.0 

3.3 
2.6 

4.9 
54.0 

47.0 
26.0 

0.5 
1.1 

1.2 
1.8 

1.5 
0.7 

Cottonseed  hulls,  timothy  hay 
and  white  corn 

Timothy     hay,     cottonseed 
hulls,  cottonseed   meal   and 
yellow  corn 

Timothy    hay,    cottonseed 
hulls,       cottonseed       meal, 
yellow    corn,    and    20    Ibs. 
carrots  per  day  
Timothy    hay,       cottonseed 
hulls,  cottonseed  meal,  yel- 
low corn,  and  20  Ibs.   car- 
rots per  day  

Timothy       hay,       cottonseed 
hulls,  cottonseed  meal,  and 
yellow  <~"orn 

424       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

The  above  table  shows  in  a  very  striking  manner  that  the  amount 
of  carotin  in  the  blood  serum  of  the  lactating  cow,  as  well  as  the 
amount  of  carotin  and  xanthophylls  in  the  milk  fat,  is  dependent  upon 
the  ration.  The  figures  in  the  table  also  show  very  clearly  why  the 
butter  fat  in  the  first  two  cases  was  not  absolutely  colorless.  These 
two  samples  were  taken  at  the  end  of  a  long-continued  feeding  of 
a  ration  almost  entirely  lacking  in  carotin  and  xanthophylls,  which 
was  planned  for  the  purpose  of  eliminating  as  far  as  possible  the 
color  from  the  milk  fat.  It  is  evident  that  this  was  not  accomplished 
in  the  strictest  sense  of  the  word  because  the  blood  serum  in  both 
cases  still  contained  a  small  amount  of  carotin. 

The  results  of  these  studies  were  so  striking  that  it  was  con- 
sidered advisable  to  confirm  them  if  possible  with  other  animals. 
We  fortunately  had  at  hand,  6  cows  of  3  different  breeds,  all  pure 
bred  animals,  which  were  in  ideal  condition  for  such  an  experiment. 
They  had  all  just  completed  an  experiment  in  which  their  feed  for 
12  to  14  weeks  had  been  essentially  a  non-pigmented  one,  being  made 
up  of  cottonseed  meal,  corn  stover  and  very  light-colored  timothy 
hay.  A  night  and  morning  milking  of  each  cow  was  combined 
and  after  determining  the  percentage  of  fat  in  the  combined  sample  l 
the  milk  was  separated,  the  cream  churned,  and  the  color  of  the 
rendered  butter  fat  observed  in  the  Tintometer.  The  same  day,  sam- 
ples of  blood  were  drawn  from  each  animal  and  the  color  of  the 
serum  determined  by  the  method  described  above.  The  feed  of  the 
cows  was  now  changed  so  that  it  was  largely  made  up  of  alfalfa 
hay,  rich  in  carotin  and  xanthophylls,  and  later  a  little  fresh  green 
pasture  grass.  Thirty  days  after  the  cows  had  been  on  this  feed 
the  first  experiment  was  repeated  and  the  color  of  the  butter  fat 
and  blood  serum  again  observed.  The  results  of  this  experiment 
are  given  in  Table  No.  4. 

1.     The  weight  of  the  combined  sample  was  also  recorded. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  425 


TABLE   No.    4. — RELATION   BETWEEN   CHARACTER   OF   RATION   AND   AMOUNT   OF 
PIGMENT  IN  MILK  FAT  AND  BLOOD  SERUM. 


Cow 

No. 

Breed 

Date 

Grams 
fat. 

Butterfat 

Serum 

Yellow 

Red 

Yellow 

Red 

213 
213 

Holstein 
Holstein 

3-11-13 
4-10-13 

122 
135 

8.5 
54.0 

1.4 
1.8 

6.0 
48.0 

0.7 
1.1 

220       JHolstein 
220       iHolstein 

I 

3-11-13 
4-10-13 

167 
208 

3.0 
22.0 

0.7 
1.2 

7.0 
41.0 

0.8 
1.0 

303        Ayrshire 
303        (Ayrshire 

3-11-13 
4-10-13 

213 
263 

2.5 
16.0 

0.6 
1.1 

11.0 
40.0 

0.9 
1.0 

16 
16 

Jersey 
Jersey 

3-11-13 
4-10-13 

304 

363 

11.0 
64.0 

1.7 
2.0 

10.0 
45.0 

0.9 
1.1 

57 
57 

Jersey 
Jersey 

3-11-13 
4-10-43 

240 
263 

5.2 
54.0 

1.2 
1.7 

13.0 
57.0 

1.1 
1.8 

64 
64 

Jersey 
Jersey 

3-11-13 
4-10-13 

281 
350 

4.7 
47.0 

1.5 
1.6 

7.5 
45.0 

0.7 
1.0 

The  results  of  this  experiment  are  as  striking  and  conclusive 
as  in  the  experiment  with  Cow  No.  301,  and  show  that  there  is  a 
direct  relation  between  the  amount  of  carotin  in  the  food  and  the 
amount  of  lutein  in  the  blood  serum,  just  as  there  is  a  direct  relation 
between  the  presence  of  an  excess  of  carotin  in  the  food  and  the 
production  of  a  high-colored  butter  fat.  It  is  necessarily  true  also 
that  there  is  a  direct  relation  between  the  color  of  the  butter  fat 
and  the  amount  of  lutein  in  the  blood  serum.  A  small  amount  of 
lutein  in  the  blood  serum  will  always  mean  a  light-colored  butter  fat. 
It  does  not  appear  to  be  necessarily  true,  however,  that  a  high-colored 
serum  will  be  accompanied  by  a  high-colored  butter  fat.  The  only 
conclusion  in  this  connection  that  can  be  drawn  from  the  data  of  Tables 
3  and  4  is  that  an  increase  in  the  color  of  the  blood  is  accompanied  by 
an  increase  in  the  color  of  the  fat.  The  actual  color  of  the  fat  under 
these  conditions  is  apparently  dependent  upon  a  number  of  conditions 
which  are  not  explained  by  this  data.  The  amount  of  fat  being 
produced  and  the  breed  of  the  animal  are  both  factors  which  probably 
influence  the  color  of  the  fat.  There  is  certainly  a  wider  difference 


426      MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

between  the  color  readings  of  the  butter  fats  than  between  the  color 
readings  of  the  blood  serum  extracts  during  the  second  part  of  the 
experiment  whose  data  are  given  in  Table  No.  4. 

It  is  possible  that  this  difference  may  be  explained  on  the  ground 
that  the  albumin  content  of  the  milk  is  in  some  way  closely  related 
to  the  color  of  the  milk  fat.  At  any  rate  the  data  given  in  the 
following  table  admit  of  such  interpretation.  The  conditions  of 
the  animal  preceding  the  data  were  as  follows :  Cow  No.  301  had 
been  subjected  to  severe  underfeeding,  i.  e.,  she  received  only  about 
70  per  cent  of  the  food  required  to  produce  her  milk  and  maintain 
her  body  weight.  The  food  she  received  during  this  time  was  com- 
posed of  about  5  pounds  of  a  mixture  of  corn,  bran  and  linseed  meal, 
and  about  7  pounds  of  alfalfa  hay,  a  ration  moderately  rich  in  carotin. 
Her  ration  was  then  changed  to  one  practically  free  from  carotin, 
consisting  of  white  corn,  cottonseed  meal  and  bleached  alfalfa  hay. 
The  cow  was  brought  back  to  a  normal  plane  of  nutrition  on  this 
ration.  The  immediate  effect  on  the  composition  of  the  milk  and 
the  color  of  the  milk  fat  is  shown  in  Table  5.  The  subsequent 
effect  upon  the  color  of  the  milk  fat  is  given  in  Table  No.  15  of 
the  preceding  Bulletin  of  this  series  which  dealt  with  the  milk  fat 
pigment. 

TABLE  No.  5. — A  POSSIBLE  RELATION  BETWEEN  THE  ALBUMIN  OF  MILK  AND  THE 
COLOR  OF  THE  MILK  FAT.  * 
AYRSHIRE  Cow  No.  301. 


Milk   fat 

Total 

Casein 

Albu-     Color     of     fat 

Date 

Hay 

Grain 

per  day 

protein 

min      ' 

1912 

Lbs. 

Lbs. 

Grams 

Grams 

Grams 

Grams    Yellow 

Red 

(c)       i 

9-23-24 

7.2fa) 

4.9 

254 

209 

138 

37            15 

1.8 

9-25-26 

8.  Kb) 

4.9 

249 

203 

134 

36 

15 

1.8 

9-27-28 

11.0 

6.0 

262 

222 

138 

47 

19 

1.8 

9-29-30 

14.0 

7.0 

272 

230 

135 

57 

21 

1.7 

10-1-2 

14.5 

7.5 

267 

237 

132 

67 

28 

1.8 

10-3-4 

9.8 

8.0 

276 

220 

139 

46 

20 

1.7 

(a)  Alfalfa  hay,  rich  in  carotin. 

(b)  Bleached  alfalfa  hay,  free  from  carotin. 

(c)  Calculated. 


The  data  in  Table  No.  5  shows  that  although  the  conditions  of 
feed  were  such  that  a  decline  in  color  would  be  expected,  the  reverse 
was  found.  The  point  to  be  emphasized  is  that  the  sharp  increase 
in  the  color  of  the  milk  fat  was  coincident  with  an  increase  in  the 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT 


427 


albumin  content  of  the  milk.  The  result  points  to  the  probabilit) 
of  a  relation  between  the  higher  color  and  the  increase  in  albumin. 
Additional  evidence  pointing  to  the  same  relation  is  the  presence  of 
extremely  high  color  along  with  a  very  abnormal  amount  of  albumin 
in  colostrum  milk,  as  pointed  out  later  in  this  paper. 

An  attempt  was  made  to  determine  if  any  definite  relation 
exists  between  the  albumin  and  color  as  found  in  the  milk  of 
various  individuals.  The  milk  of  12  cows  representing  the  Jersey, 
Holstein  and  Ayrshire  breeds  was  used.  The  feed  received  was 
pasture  grass  and  some  grain.  The  color  of  the  milk  fat  and  the 
percentage  of  albumin  were  determined  for  each  animal.  The  results 
of  the  study  are  given  in  Table  No.  6. 

TABLE  No.  6. — RELATION  BETWEEN  THE  AI/BUMIN  CONTENT  OF  MILK  AND  THE 
COLOR  OF  MILK  FAT,  UNDER  NORMAL  CONDITIONS. 


Pounds 

Grams 

Grams 

Grams 

Color  of  fat. 

Breed 

Cow  No. 

milk 

protein 

albumin 

fat 

per 

per  day 

per  day 

per  day 

day 

Yellow 

Red 

j 

14 

18.4 

311.0 

24.45 

459.0 

64.0 

2.7 

j 

64 

12.9 

254.0 

22.65 

380.2 

64.0 

2.8 

j 

57 

10.8 

202.0 

22.02 

267.0 

64.0 

3.0 

A. 

303 

20.2 

310.0 

33.20 

363.5 

50.0 

1.7 

H. 

221 

15.1 

270.0 

27.05 

257.0 

43.0 

1.8 

H. 

222 

14.5 

233.8 

20.12 

202.5 

33.0 

1.3 

j\ 

16 

20.8 

339.0 

35.75 

414.0 

64.0 

3.1 

j. 

317 

13.0 

234.5 

41.00 

259.0 

80.0 

3.5 

H. 

225 

13.3 

200.0 

22.35 

150.9 

47.0 

1.8 

H! 

220 

16.3 

525.0 

35.17 

192.5 

24.0 

1.7 

A. 

301 

18.7 

282.5 

39.00 

273.0 

54.0 

2.0 

H. 

213 

11.4 

161.5 

19.05 

144.2 

64.0 

2.7 

"J."  stands  for  Jersey,  "H."  for  Holstein,  "A."  for  Ayrshire. 

There  appears  to  have  been  no  relation  between  the  albumin  of 
the  milk  and  the  color  of  the  milk  fat  of  these  animals.  It  is  not 
considered  however,  that  these  results  are  conclusive  either  as  prov- 
ing or  disproving  the  supposition  that  such  a  relation  exists.  Our 
knowledge  of  the  subject  is  too  limited  at  present  to  enable  us  to 
control  all  the  factors  that  enter  into  the  question. 

THE  TRANSPORTATION  OF  CAROTIN  AND  XANTHOPHYLLS  BY  THE 

BLOOD  SERUM. 

When  it  had  been  shown  conclusively  that  the  lutein  of  the 
blood  serum  of  the  cow  is  composed  of  the  carotin  and  xanthophyll 
pigments  of  the  food,  taken  up  along  the  digestive  tract  and  transmitted 
by  means  of  the  blood  to  the  fat  synthesizing  cells  of  the  milk  glands 


428       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

and  body  tissue,  it  became  a  matter  of  considerable  importance  to 
ascertain  how  the  blood  carries  these  pigments  through  the  body. 
It  has  already  been  shown  that  there  is  a  strong  possibility  that  the 
minor  constituent  of  the  pigment,  i.e.,  the  xanthophylls,  are  carried 
in  the  serum  dissolved  in  the  fat.  When  considering  such  physiological 
phenomena  as  the  great  volume  of  blood  in  circulation  in  an  animal 
as  large  as  the  cow  and  the  rapidity  with  which  it  circulates,  it 
would  seem  very  probable  that  even  the  very  small  percentage  of 
fat  in  the  blood  is  sufficient  to  account  for  all  the  pigment,  both 
carotin  and  xanthophylls,  which  is  presented  to  the  milk  glands  and 
body  cells.  On  the  other  hand,  when  one  considers  the  very  large 
proportion  of  carotin  which  is  present  in  any  given  quantity  of  blood 
serum  of  a  cow  receiving  a  ration  rich  in  carotin,  it  must  be  concluded 
that  the  fat  plays  little  if  any  part  in  the  transportation  of  this 
pigment.  The  studies  that  are  to  be  presented  will,  therefore,  consider 
only  the  carotin  of  the  blood  serum,  since  it  is  this  pigment  that  com- 
prises by  far  the  greatest  proportion  of  the  lutein  of  the  serum. 

It  might  be  considered  that  the  carotin  is  carried  by  the  serum 
merely  in  simple  solution.  In  fact,  Thudichum  l  stated  that  the  lutein 
of  the  blood  is  in  solution  in  the  serum.  This  seems  to  be  very 
probable  especially  in  view  of  the  fact  that  Krukenberg2  found  that 
it  could  be  extracted  from  the  serum  by  means  of  amyl  alcohol. 

Many  facts  can  be  presented,  however,  that  go  to  show  that 
the  carotin  does  not  exist  in  the  serum  in  simple  solution.  In  the 
first  place  neither  carotin  from  plants  nor  the  carotin  of  the  serum 
itself  are,  when  isolated,  taken  up  to  any  extent  when  treated  with 
the  pure  blood  serum.  Blood  serum  almost  free  from  carotin  from 
natural  causes,  showed  no  indication  of  having  taken  up  the  carotin 
in  either  case  when  poured  over  the  pure  amorphus  pigments ;  and  the 
serum  itself  showed  no  increase  in  the  amount  of  color  that  could  be 
extracted  by  petroleum  ether  after  dessication  with  plaster  of  Paris 
and  moistening  with  alcohol. 

In  addition  to  the  above  the  following  observations  were  made : 3 

I.  Five  c.c.  portions  were  shaken  vigorously  with  equal  volumes 
of  petroleum  ether,  ether,  CS2  and  amyl  alcohol  respectively.  All 
extracts  were  colorless  except  in  the  case  of  the  amyl  alcohol  which 
was  golden-yellow,  and  showed  the  carotin  absorption  bands  both 

1.  Loc.  cit 

2.  Loc.  cit. 

3.  Except  where   stated   most  of  work  about  to  be   reported  was   done 
with  a  golden-yellow,  high-colored  serum  from  Ayrshire  cow  No.  301.     The 
serum  was  obtained  from  this  cow  by  drawing  250  c.c.  of  blood  from  the 
jugular  vein  and  allowing  it  to  clot  and  the  serum  to  press  out.     The  serum 
was  free  from  red  corpuscles. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  429 

in  carbon  bisulphide  and  alcohol,  the  solution  in  the  first  solvent 
showing  three  distinct  bands.  This  shows  that  the  lipochrome  which 
Krukenberg  extracted  from  ox  serum  was  the  pigment  which  we 
have  identified  as  carotin.  On  addition  of  alcohol  to  the  other  mixtures 
the  solvents  in  each  case  completely  extracted  the  pigment  on  shaking. 

2.  Five  c.c.  portions  were  dessicated  with  plaster  of  Paris  and 
shaken  with  ether,  petroleum  ether,  and  carbon  bisulphide,  respectively. 
A  mere  trace  of  color  was  extracted  in  each  case.     When   a  little 
absolute  alcohol  was  added  however,  in  all  cases  the  solvents  became 
well  colored. 

3.  Five  c.c.  of  serum  was  diluted  with  twenty  volumes  of  water, 
without  causing  any  precipitation  of  the  pigment. 

4.  Twenty-five  c.c.  of  serum  was  treated  with  successive  por- 
tions of  saturated    (NH4)2   SO4  solution  to  the   following  per  cent 
saturations  :  28-35,  36-40,  45-46,  and  finally  to  one-half  saturation.    The 
fractionally  precipitated  globulins  were  in  every  case  practically  free 
from  carotin,  and  the  half  saturated  globulin  free  serum  was  golden- 
yellow.     The  color  was  entirely  precipitated  from  a  portion  of  this 
solution  on  complete  saturation  with    (NH4)2   SO4.     The  remainder 
was  acidified  with  a  few  drops  of  i^  per  cent  acetic  acid  and  heated 
to  about  80 °C.     The  coagulated  albumins  carried  down  only  a  small 
part   of   the  color.     The   entire   pigment   was   precipitated    from   the 
filtrate,  however,  on  complete  saturation  with   (NH4)2  SO4  in  sub- 
stance, the  light  precipitate  which  came  down  being  deep  yellow  in 
color.     This   deep   yellow   precipitate   was    readily   soluble   in   water 
giving  a  clear  yellow  aqueous  solution  from  which  neither  ether  nor 
petroleum  ether  would  extract  any  color  until  the  protein  in  the  solu- 
tion had  first  been  coagulated  with  alcohol. 

5.  Five  c.c.  of  serum  was  diluted  to  25  c.c.  with  distilled  water 
and  the  solution  saturated  with  Mg  SO4  in  substance.     The  globulins 
were  filtered  off.  The  filtrate  was  golden-yellow.  Acetic  acid  was  added 
to  a  concentration  of   I   per  cent.     The  precipitated   albumins   were 
bright  yellow,   leaving  the   solution   colorless.     Petroleum   ether   and 
carbon  bisulphide  extracted  a  slight  amount  of  color  from  this  pre- 
cipitate on  long  contact.     After  the  addition  of  a  little  alcohol,  how- 
ever, both  solvents  readily  extracted  the  color. 

6.  One  hundred  c.c.  of  serum   from  Jersey  Cow  No.  25  was 
diluted  with  several  volumes  of  water,  a  pinch  of  NaCl  and  a  few 
drops  of  glacial  acetic  acid  added  and  the  solution  heated  quickly  to  a 
temperature  just  below  the  boiling  point.    The  coagulum  which  formed 
contained  a  very  little  pigment  but  the  filtrate  was  golden-yellow.     No 
color  could  be  extracted  from  the  filtrate  by  carbon  bisulphide,  or  by 


43°       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

ether,  even  after  making  strongly  alkaline  with  potassium  hydroxide. 
Amyl  alcohol  extracted  the  pigment. 

7.  Experiment  6  repeated  on  200  c.c.  of  the  same  serum  gave  the 
same  result.     The  entire  pigment   in  the  golden-yellow   nitrate  was 
coagulated  by  boiling.    The  coagulum  was  not  soluble  in  water. 

8.  Fifty  c.c.  of  serum  (Cow  No.  2)  was  diluted  to  350  c.c.  with 
water,  a  pinch  of  salt  added  and  the  solution  heated  on  the  steam 
bath,  with  stirring  until  cloudiness  appeared.     On  adding  a  few  drops 
of  glacial  acetic  acid,  a  sharp  coagulation  took  place.     On  filtering, 
the  filtrate  was  bright  yellow  in  color.     On  saturation  of  the  filtrate 
with  (NH4)2  SO4  in  substance,  a  comparatively  small  amount  of  deep 
yellow  precipitate  was  thrown  down  leaving  a  colorless  supernatant 
solution.    The  yellow  precipitate,  which  was  contaminated  with  a  little 
(NH4)2  SO4,  was  readily  soluble  in  water,  giving  a  perfectly  clear 
yellow  solution  from  which  the  yellow  color  was  again  entirely  thrown 
down  on  saturation  with  (NH4)2  SO4  in  substance,  or  on  the  addition 
of  mercuric  nitrate.     The  latter  precipitate  when  still  moist  would  not 
give  up  its  color  to  petroleum  ether  until  first  moistened  with  absolute 
alcohol.     The  bright  yellow  pigment  now  found  in  the  petroleum  ether 
gave    a    red-orange    CS2    solution   which    showed    the    three    carotin 
absorption  bands. 

9.  Two  350  c.c.  portions  of  serum   (Jersey  Cow  No.  2)   were 
treated  respectively  as  follows : 

Portion  A  was  treated  with  (NH4)2  SO4  in  substance  to  one  half 
saturation,  according  to  the  formula 

VC2 
X=     


18.158  — .54  C2 

Where  V  —  original  volume  of  protein  solution. 
C  =  desired  saturation  as  grams  in  10  c.c. 
X  =  grams  to  be  added  to  give  the  required 
saturation. 

The  globulins  which  precipitated  carried  down  some  of  the  pig- 
ment, but  on  dissolving  them  in  150  c.c.  of  warm  water  containing  some 
(NH4)2  SO4,  and  adding  (NH4)2  SO4  to  half  saturation,  they  were 
thrown  down  practically  colorless.  The  yellow  filtrate  from  this  pre- 
cipitation was  added  to  the  other  globulin-free  filtrate  and  the  com- 
bined solutions  diluted  to  1500  c.c.  with  distilled  water.  This  solution 
was  now  raised  to  a  temperature  of  75 °C  in  a  water  bath.  15  c.c.  of 
i%  per  cent  acetic  acid  added  and  the  temperature  raised  to  80  °C, 
when  a  sharp  coagulation  occurred.  The  solution  was  filtered,  giving  a 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  43! 

perfectly  clear  bright  yellow  filtrate.  It  was  saturated  with  (NH4),>. 
SO4  in  substance,  throwing  down  a  small  amount  of  deep  yellow 
precipitate.  The  precipitate  was  filtered  off  on  a  large  (n-inch) 
Biichner  funnel,  using  suction,  so  that  the  layer  of  yellow  protein 
would  be  as  free  as  possible  from  occluded  (NH.4)2  SO4.  The  golden- 
yellow  precipitate  was  sucked  as  dry  as  possible  on  the  funnel  and  the 
sticky  mass  covering  the  paper  in  very  thin  layer  dissolved  in  warm 
water,  in  which  it  was  readily  soluble,  and  the  clear  yellow  solution 
set  aside. 

Portion  B  was  diluted  with  an  equal  volume  of  distilled  water, 
a  little  sodium  chloride  added,  and  the  solution  raised  to  a  temperature 
of  75  °C.  in  a  water  bath.  Acetic  acid  was  now  added  carefully  until 
a  heavy  definite  coagulation  took  place.  The  coagulated  proteins  car- 
ried down  some  of  the  pigment  but  by  far  the  greatest  part  was  in  the 
clear  yellow  filtrate.  This  filtrate  was  saturated  with  (NH4)2  SO4 
in  substance  and  the  precipitated  pigmented  protein  filtered  off  in  the 
same  way  as  in  the  case  of  portion  A.  After  being  made  comparatively 
dry  by  suction,  the  deep  yellow  residue  was  readily  soluble  in  a  small 
amount  of  cold  distilled  water. 

The  two  similar  solutions  from  A  and  B  were  now  combined  and 
filtered  on  a  small  Biichner  funnel  through  several  layers  of  fine  paper 
to  free  it  from  dirt  and  other  foreign  matter  introduced  by  the  (NHt)2 
SO4.  The  golden-brownish-yellow  filtrate  of  about  250  c.c.  volume 
had  a  faint  cloudiness  when  viewed  by  transmitted  light  and  contained 
some  (NH4)2  SO4.  That  the  pigment  of  this  solution  was  carotin  was 
shown  by  the  fact  that  when  an  equal  volume  of  alcohol  was  added  to 
15  c.c.  of  the  solution  and  the  mixture  was  shaken  with  petroleum 
ether,  the  petroleum  ether  rose  to  the  top  as  a  golden-yellow  solution, 
leaving  the  lower  cloudy  alcoholic  layer  colorless.  The  pigment  in 
the  petroleum  ether  layer  gave  a  red-orange  carbon  bisulphide  solution, 
and  in  this  solvent  showed  the  usual  carotin  absorption  bands. 

The  aqueous  solution  was  now  dialysed  in  a  parchment  bag 
against  running  water  for  eight  days.  At  the  end  of  this  time  the 
solution  was  still  giving  a  precipitate  with  barium  chloride  indicating 
that  the  solution  was  not  free  from  (NH4)2  SO4.  No  protein  crystal- 
lization had  occurred,  but  decomposition  had  begun,  for  the  solution 
was  cloudy,  and  showed  a  very  fine  coagulation.  This  coagulum  was 
filtered  off.  It  had  a  dirty  brown  color  and  when  almost  dry  was  quite 
sticky.  It  was  not  soluble  in  water,  but  both  in  the  dry  state  and  in 
suspension  in  water  it  gave  up  a  golden-yellow  color  to  ether,  leaving- 
the  precipitate  dirty  white  in  color.  The  extracted  pigment  showed 
the  three  absorption  bands  of  carotin  in  carbon  bisulphide  solution.. 


432       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

In  the  solid  state  the  pigment  was  insoluble  in  absolute  alcohol  but 
readily  soluble  in  petroleum  ether — absolute  alcohol,  from  which  the 
petroleum  ether  readily  extracted  it  on  dilution  with  a  little  water. 
The  solid  pigment  was  also  very  difficultly  soluble  in  alcoholic  potash. 
After  saponification  for  one  half  hour  and  extraction  by  ether,  the 
pigment,  in  the  solid  state,  was  fused  with  a  little  solid  sodium 
hydroxide  and  potassium  nitrate  in  a  porcelain  crucible.  The  flux  was 
dissolved  in  hot  water  and  the  solution  evaporated  to  dryness  on  the 
steam  bath  in  the  presence  of  an  excess  of  c.  p.  HNO3.  As  much  as 
possible  of  the  residue  was  dissolved  in  hot  water  containing  some  c, 
p.  HNOS,  the  solution  filtered  and  an  equal  volume  of  pure  molybdate 
solution  added  to  the  100  c.c.  of  filtrate.  On  digestion  at  60° C,  for 
several  hours  there  was  a  distinct  yellow  precipitate  of  ammonium 
phosphomolybdate. 

Returning  to  the  slightly  cloudy  but  yellow  aqueous  filtrate  from 
the  dialysed  solution,  we  found  that  the  color  could  be  entirely  thrown 
down;  (i)  by  acid  lead  acetate  as  a  light  yellow  precipitate  which 
bleached  almost  entirely  in  12  hours,  but  from  which  petroleum  ether 
extracted  a  faint  yellow  color  after  soaking  in  alcohol  for  about  one 
hour;  (2)  by  nitric  acid  mercuric  nitrate  solution  as  a  bright  yellow 
precipitate  which  was  very  stable  and  gave  up  its  color  to  petroleum 
ether  only  after  soaking  in  alcohol; 1  (3)  by  neutral  ten  per  cent  solu- 
tion of  AgNO3  as  a  deep  yellow  precipitate  which  was  stable  although 
darkening  badly  as  the  AgNO3  oxidized  in  the  light,  but  readily  giving 
up  its  color  to  petroleum  ether  on  addition  of  alcohol  to  the  precipitate, 
the  pigment  thus  extracted  showing  the  carotin  bands  in  CS2  solution ; 
(4)  on  saturation  with  (NH4)2  SO4  in  substance  as  a  deep  yellow 
precipitate  which  was  not  soluble  in  water  but  when  suspended  in  water 
gave  up  its  color  to  petroleum  ether  only  after  the  addition  of  abso- 
lute alcohol;  (5)  on  addition  of  an  excess  of  alcohol  as  a  yellow  pre- 
cipitate which  when  dry  gave  up  no  color  to  petroleum  ether  alone, 
but  to  alcoholic  petroleum  ether  gave  up  a  yellow  pigment  which  was 
quantitatively  found  in  the  petroleum  ether  on  separation  of  the  alco- 
hol with  a  little  water;  (6)  on  heating  the  neutral  solution  to  boiling 
as  a  yellow  coagulum  insoluble  in  water  and  giving  up  no  color  to  hot 
alcohol  or  petroleum  ether. 

In  addition  to  the  above  observations  the  following  may  be  men- 
tioned. In  working  with  a  large  number  of  samples  of  blood  serum 

1.  The  pigment  thus  extracted  showed  the  three  carotin  absorption  bands 
in  carbon  bisulphide  solution;  in  alcoholic  solution  it  gave  a.  pronounced 
precipitate  of  digitonin-cholesteride  on  addition  of  hot  one  per  cent  digitonin 
solution  in  ninety  per  cent  alcohol. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  433 

it  was  often  noticed  that  when  the  serum  had  stood  for  some  time 
in  a  closed  bottle  in  contact  with  a  little  supernatant  air,  an  orange- 
yellow  scum  always  came  to  the  top.  This  was  found  to  be  a  water 
insoluble  protein  which  would  not  give  up  any  color  to  petroleum 
ether  when  its  aqueous  suspension  was  shaken  with  that  solvent,  but 
when  an  equal  volume  of  absolute  alcohol  was  added  and  the  shaking 
with  petroleum  ether  repeated,  the  latter  solvent  rose  to  the  top  as  a 
beautiful  yellow  layer.  The  pigment  thus  extracted  gave  a  red-orange 
carbon  bisulphide  solution  showing  the  three  carotin  absorption  bands. 

In  order  to  show  more  conclusively  the  character  of  the  protein 
with  which  the  serum  carotin  is  evidently  combined,  the  coagulation 
temperature  of  the  protein  was  determined.  For  this  purpose  150  c.c. 
of  serum  was  diluted  with  an  equal  volume  of  a  saturated  solution  of 
ammonium  sulphate.  After  filtering  off  the  precipitated  globulins,  por- 
tions of  the  globulin-free  filtrate  were  submitted  to  fractional  coagu- 
lation. It  was  found  that  on  carefully  elevating  the  temperature  to 
80°  C.  and  holding  it  at  that  temperature  for  a  short  time,  the  filtrate 
from  the  coagulated  albumins  still  yielded  a  large  amount  of  carotin  on 
addition  of  alcohol  and  shaking  with  petroleum  ether.  On  the  other 
hand  the  coagulated  albumins  yielded  a  comparatively  small  amount 
of  carotin.  A  similar  result  was  obtained  at  temperatures  of  81°,  82°, 
83°,  84°,  85°,  and  85.5°  C.,  although  the  amount  of  carotin  in  the 
filtrate  rapidly  decreased  with  the  increase  in  coagulation  temperature. 
At  86°  C.,  however,  the  pigmented  protein  had  completely  coagulated, 
and  the  filtrate  yielded  no  carotin  on  treatment  with  alcohol  and 
petroleum  ether.  The  coagulation  temperature  limits  of  the  pigment 
carrying  protein  therefore  lie  between  80°  and  86°  C.,  when  the  pro- 
tein is  in  half  saturated  ammonium  sulphate  solution.  There  is  no 
marked  coagulation  at  the  lower  temperature,  but  it  is  completely 
coagulated  at  the  upper  temperature. 

The  coagulation  temperature  of  the  protein  which  carries  the 
carotin  in  the  blood  was  studied  further  with  an  aqueous  solution  of 
the  protein  obtained  in  a  manner  similar  to  the  one  used  in  obtaining 
the  protein  for  the  study  previously  reported.  Briefly,  an  equal  volume 
of  saturated  (NH4)2  SO4  solution  was  added  to  200  cubic  centimeters 
of  blood,  rich  in  carotin,  from  Holstein  Cow  No.  221.  The  globulins 
were  filtered  off  and  the  golden-yellow  filtrate  heated  carefully  in  a 
water  bath  to  a  temperature  of  79°  C.  The  coagulated  proteins  were 
filtered  off.  The  yellow  filtrate  was  saturated  with  (NH4)2  SO4 
in  substance  and  let  stand  several  hours.  The  golden-yellow  pre- 
cipitate was  filtered  off  on  a  Buchner  funnel.  After  allowing  to  suck 


434       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

quite  dry,  all  crystals  of  (NH4)2  SO4  were  removed  with  a  spatula 
and  the  protein  dissolved  in  about  75  cubic  centimeters  of  water,  in 
which  it  was  readily  soluble.  This  deep  yellow  solution  was  neutral 
in  reaction.  It  contained  a  small  amount  of  (NH4)2  SO4,  the  amount 
of  which  was  determined  quantitatively.  (NH4)2  SO4  was  added 
to  a  portion  of  this  solution  to  bring  the  concentration  up  to  a  normal 
solution.  An  equal  volume  of  saturated  (NHt)2  SO.,  solution  was 
added  to  the  remainder,  giving  a  solution  between  3  and  4  normal  and 
one  similar  to  the  one  whose  coagulation  temperature  was  observed 
above.  The  coagulation  temperature  of  the  pigmented  protein  of  these 
two  solutions  was  carefully  studied.  Both  solutions  were  found  to 
contain  a  small  amount  of  colorless  protein  which  coagulated  between 
65°  and  75°  C.  This  was  filtered  off,  the  nitrate  retaining  its  original 
yellow  color.  This  nitrate  was  then  studied  further. 

The  3-4  normal  or  one  half  saturated  (NH4)2  SO4  solution  acted 
in  a  manner  identical  with  the  solution  whose  study  is  recorded  above. 
The  first  opalescence  appeared  between  79°  and  80°  C.,  and  complete 
coagulation  did  not  take  place  until  the  temperature  was  raised  to 
86°  C. 

It  was  not  found  possible  to  cause  a  clear  coagulation  of  the 
pigmented  protein  in  the  neutral  normal  solution  of  (NH4)..  SO4  even 
when  the  temperature  was  raised  to  90°  C.  Opalescence  began,  how- 
ever, between  82°  and  82.5°  C.  Coagulation  was  readily  obtained 
when  the  solution  was  heated  to  89°  C.  in  the  presence  of  a  very  little 
HC1.  (3  drops  of  a  */2  normal  HC1  solution  were  added  to  10  c.  c.  of 
solution.) 

The  large  amount  of  evidence  which  has  now  been  submitted  in 
regard  to  the  transportation  of  the  carotin  in  the  blood  serum  will 
justify  but  one  conclusion,  namely  that  the  carotin  exists  in  the  blood 
in  conjugation  with  one  of  the  proteins.  The  evidence  will  also  justify 
the  conclusion  that  the  protein  with  which  the  carotin  is  combined  is 
an  albumin. 

Summarizing  the  evidence,  we  have  shown  that  the  carotin  car- 
rying protein  is  precipitated  from  its  solution  in  the  serum  or  from  its 
aqueous  solution,  on  complete  saturation  only  with  ammonium  sulphate, 
or  by  saturation  with  magnesium  sulphate  only  in  one  per  cent  acetic 
acid  solution,  or  by  heating  its  half  saturated  ammonium  sulphate 
solution  to  86°  C. ;  the  protein  may  also  be  coagulated  by  alcohol,  or 
by  boilding  its  solution  in  the  presence  of  acetic  acid.  As  in  all  salting 
out  methods  for  the  precipitation  of  proteins,  the  pigmented  protein 
is  readily  soluble  in  water  after  being  thrown  down  by  ammonium 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  435 

sulphate,  but  is  no  longer  soluble  in  water  after  being  coagulated  by 
heat  or  alcohol.1  The  protein  is  not  coagulated  by  saturation  of  its 
solution  with  sodium  chloride. 

Not  very  much  can  be  said  in  regard  to  the  character  of  the 
combination  of  the  carotin  with  the  albumin  The  combination  is  evi- 
dently a  firm  one,  and  is  broken  down  only  in  the  presence  of  alcohol 
so  that  the  pigment  can  be  extracted  by  ether  or  petroleum  ether.  The 
union  is  also  broken  down  by  dialysis,  or  at  least  rendered  less  firm, 
but  is  not  broken  down  when  the  protein  is  precipitated  as  a  lead,  silver 
or  mercury  salt.  It  is  interesting  to  notice  that  cholesterol  and  a  phos- 
phorus-containing body  (probably  lecithin)  are  mixed  up  in  some  way 
in  the  combination  of  albumin  and  carotin,  the  liberated  carotin  from 
the  dialysed  pigmented  albumin  yielding  both  cholesterol  and  phospho- 
rus. We  propose  the  name  caroto-albumm  or  luteo-albumin  for  the 
chromo-protein  which  transmits  the  carotin  from  the  food  to  the  milk 
glands  and  fat  synthesizing  body  cells  of  the  cow. 

The  finding  of  this  highly  unsaturated  hydrocarbon  carotin  pig- 
ment in  combination  with  one  of  the  albumins  of  the  blood,  probably 
similar  to  the  combination  of  the  haematin  in  the  haemoglobin  of  the 
red  blood  corpuscles,  at  once  raises  some  impiortant  questions  as  to 
a  possible  physiological  significance  which  might  be  attached  to  the 
presence  of  the  pigment.  One  can  only  suggest  that  like  the  haemo- 
globin the  luteo-albumin  may  be  of  importance  in  connection  with  the 
oxygen  supply  of  the  body.  This  is  not  probable.  The  ease  with  which 
the  carotin  is  increased  and  decreased  in  the  blood  serum  as  shown  by 
the  feeding  experiments,  seems  to  preclude  the  possibility  of  the 
carotin  being  absolutely  essential  to  the  life  of  the  cow. 

A  STUDY  OF  THE  HIGH  COLOR  OF  COLOSTRUM   MILK  FAT. 

Considerable  data  was  given  in  the  preceding  Bulletin  of  this  series, 
which  showed  that  colostrum  milk  fat  from  all  breeds  of  cows  is 
characterized  by  a  very  high  content  of  carotin.  In  view  of  the  results 
obtained  in  the  study  of  the  pigment  of  the  blood  serum,  it  seemed 
very  probable  that  this  interesting  phenomenon  was  due  to  a  great 
accumulation  of  the  carotin  in  the  blood  serum  just  previous  to  par- 
turition. In  order  to  obtain  some  definite  experimental  evidence  in 
support  of  this  supposition,  blood  was  drawn  from  the  jugular  vein  of 
a  pure  bred  Jersey  cow  (No.  23),  when  she  was  dry  and  three  days 
previous  to  parturition.  The  amount  of  color  in  10  c.c.  of  the  blood 

1.     After  standing  a  short  time  under  the  alcohol. 


436      MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 


was  determined  in  the  manner  previously  described.  Thirty  days 
after  calving  the  blood  was  tested  again.  The  color  of  the  milk  fat 
was  determined  at  this  time  also.  The  color  of  the  blood  serum  of 
another  Jersey  cow  was  determined  at  another  time  twelve  hours 
previous  to  the  time  it  was  judged  she  would  give  birth  to  a  calf.  The 
data  obtained  in  the  two  experiments  are  given  in  Table  7. 

TABLE  No.  7. — RELATION  OF  BLOOD  SEBUM  COLOB  TO  COLOB  OF  COLOSTRUM 

MILK  FAT. 


Remarks. 

Color  of 
serum 

Color  of 
fat. 

Yellow 

Red 

Yellow 

Red 

Three  days  before  parturition  (Jersey 
Cow  No.  23.)  ..... 

42.0 
50.0 
29.0 

0.8 
2.0 
0.2 

64.0 

1.8 

Thirty  days  after  parturition  (Jersey 
Cow  No.  23.).. 

Twelve  hours  before  parturition  (Jersey 
Cow  No.  2.)  

It  is  readily  seen  that  another  explanation  must  be  sought  for 
the  high  color  of  colostrum  milk  fat,  other  than  an  accumulation  of 
carotin  in  the  blood.  No  doubt  a  certain  amount  of  storing  up  of 
carotin  does  occur  if  a  cow  is  dry  previous  to  parturition  and  the 
serum  is  low  in  color  at  the  time  of  drying  up,  it  being  supposed 
of  course  that  the  food  contains  a  plentiful  supply  of  carotin.  The 
data  presented  in  Table  7,  when  coupled  with  the  data  in  Tables  3 
and  4,  show  very  clearly  that  under  normal  conditions  the  amount 
of  pigment  carried  by  the  serum  does  not  exceed  a  certain  maximum 
point  that  appears  to  be  practically  the  same  for  all  cows,  regardless 
of  breed.  This  is  not  abnormal  when  it  is  considered  that  the  carotin 
of  the  blood  serum  is  in  combination  with  a  protein  which  no  doubt 
comprises  a  more  or  less  constant  proportion  of  the  blood. 

This  result  forces  us  to  the  same  conclusion  reached  in  connection 
with  the  study  of  the  physiological  relation  between  food,  blood  serum 
and  milk  fat  carotin,  namely  that  other  factors,  among  which  may 
be  the  composition  of  the  milk,  must  be  taken  into  consideration  in 
explaining  the  pigmentation  of  milk  fat.  In  the  case  of  the  high 
color  of  colostrum  milk,  some  facts  stand  out  that  seem  to  have  a 
special  bearing  upon  the  phenomenon.  For  instance  it  is  a  well-known 
fact  that  the  milk  drawn  for  the  first  few  days  after  parturition  has 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  437 

a  very  abnormal  composition  which  is  characterized  by  a  very  low 
fat  percentage  and  a  very  high  protein  content,  the  largest  proportion 
of  which  is  albumin.  Unpublished  data  are  at  hand  which  show  a 
composition  of  some  colostrum  milks  of  1.3  per  cent  fat  and  over  4.5 
per  cent  albumin.  When  it  is  considered  that  the  carotin  is  carried 
by  the  blood  in  combination  with  an  albumin,  and  when  it  is  also 
taken  into  account  that  the  source  of  the  lactalbumin  is  undoubtedly, 
at  least  partially,  the  serum  albumin,  a  most  plausible  explanation 
of  the  high  color  of  colstrum  milk  fat  is  at  once  apparent.  It  is  also 
apparent  that  this  high  color  will  continue  until  the  milk  has  reached 
a  normal  composition  or  until  the  blood  supply  is  depleted.  That 
this  will  occur  regardless  of  breed  is  also  readily  explained  since  data 
show  that  the  maximum  color  of  the  blood  serum  does  not  materially 
differ  with  different  breeds. 

DISCUSSION  OF  RESULTS. 

The  results  of  the  foregoing  studies  in  regard  to  the  yellow 
lipochrome  of  the  blood  serum  of  the  cow  do  not  require  any  extended 
discussion.  Following  the  interesting  discoveries  set  forth  in  the 
preceding  papers  in  regard  to  the  nature  of  the  pigments  of  milk 
fat  and  body  fat  and  their  simple  physiological  relation  to  the  carotin 
and  xanthophylls  of  the  food  which  the  cow  receives,  it  was  not  sur- 
prising to  find  that  the  hitherto  practically  unknown  lipochrome  of 
the  blood  serum  of  the  same  animal  is  also  chiefly  carotin  and  bears 
the  same  relation  to  the  food  as  the  milk  fat  carotin.  We  are  thus 
able  to  establish  the  connecting  link  between  the  food  carotin  and 
the  carotin  of  the  milk  fat,  body  fat  carotin,  corpus  luteum,  etc.,  of 
the  cow. 

One  of  the  most  important  results  of  this  study  was  the  dis- 
covery that  the  carotin  is  not  transmitted  to  the  milk  glands  and  body 
cell  from  the  food  by  means  of  simple  solution  in  the  blood  serum, 
but  is  on  the  other  hand  carried  through  the  body  in  combination 
with  an  albumin  of  the  serum.1  This  fact  is  undoubtedly  of  con- 
siderable importance  in  connection  with  the  entire  phenomenon  of 
the  pigmentation  of  the  milk  fat.  It  may  be  safely  predicted  that 
all  the  factors  which  surround  this  phenomenon  are  in  some  way 
dependent  upon  this  fact,  and  all  these  factors  will  not  be  known  until 
it  is  clearly  understood  what  part  this  caroto-  (or  luteo-)  albumin 

1.  Incidentally  this  discovery  has  resulted  in  the  addition  of  a  new 
chromoprotein  to  the  list  of  conjugated  proteins.  This  is  itself  of  consider- 
able physiological  interest. 


MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.   12 

plays  in  the  formation  of  the  milk  fat.  The  same  holds  true  for  the 
body  fat. 

The  readily  demonstrated  fact  that  the  withdrawal  of  carotin 
from  the  food  results  in  a  marked  decrease  in  the  color  of  the  milk 
fat  being  secreted  or  in  the  body  fat  being  formed,  shows  that  the 
albumin  which  carries  the  carotin  in  the  blood  serum  does  play  a 
definite  part  in  the  formation  of  both  milk  fat l  and  body  fat  and  no 
doubt  also  in  the  formation  of  the  corpus  luteum. 

The  whole  phenomenon  offers  many  difficult  and  interesting  prob- 
lems for  future  study.  Many  of  these  when  solved  will  undoubtedly 
throw  light  upon  the  chemistry  of  the  mechanism  of  milk  secretion. 

SUMMARY. 

1.  The  well-known  lipochrome  of  the  blood  serum  of  the  cow  is, 
like  the  lipochrome  of  the  milk  fat,  body  fat,  etc.,  of  the  same  animal, 
composed  principally  of  carotin,  the  widespread  hydrocarbon  pigment 
of  plants.     Associated  in  small  quantity  with  the  carotin  of  the  serum, 
probably  dissolved  in  the  fat  of  the  blood,  are  one  or  more  xanthophyll 
pigments,  which  are  always  found  in  more  or  less  variable  quantities 
associated  with  the  carotin  of  plants. 

2.  The  carotin  and  xanthophylls  of  the  blood  serum  are  derived 
from  the  food  and  furnish  the  normal  source  for  these  pigments  in 
the  milk  fat  and  body  fat,  etc.     A  variation  in  the  quantity  of  these 
pigments  in  the  food  results  in  a  corresponding  variation  in  the  amount 
found  in  the  blood  serum  and  milk  fat.     Body  fat  formed  during  this 
time  will  be  also  affected. 

3.  The  carotin  is  carried  by  the  blood  serum  in  combination  with 
an  albumin.     The  combination  is  a  very  firm  one.     Lecithin  and  cho- 
lesterol are  probably  a  part  of  the  combination.     We  propose  the  name 
caroto-albumin  for  this  new  chromo-protein  of  the  blood. 

4.  The  caroto-albumin  of  the  blood  serum  of  the  cow  is  probably 
of  importance  in  the  formation  of  the  milk  fat,  body  fat  and  corpus 
luteum  of  the  cow.     It  is  doubtful  if  this  new  pigmented  protein  is 
of  importance  in  the  oxygen  respiration  of  the  body. 

5.  The   lactalbumin   of   cows'   milk  may,   among  other   factors, 
be  related  to  the  color  of  the  milk  fat.     There  appears  to  be  a  special 
relation  here  in  connection  with  the  high  color  and  the  high  albumin 
content  of  colostrum  milk. 

1.     The  presence  of  both  cholesterol  and  lecithin  in  the  caroto-albumin  may 
explain  the  origin  of  these  lipoids,  as  well  as  carotin  in  butter  fat. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  439 


BIBLIOGRAPHY. 

1.  Escher:     Zeit.  f.  Physiol.  Chem.  83,  p.  198  (1913). 

2.  Halliburton:     Jour.  Physiol.  7,  p.  324  (1886). 

3.  Krukenberg:     Sitz.  Ber.  d.  Jen.  Gessel.  f.  Med.  (1885). 

4.  Palmer  and  Eckles  :     Missouri  Agri.  Exp.  Sta.  Research  Bulletins 
Nos.  10,  and  n   (1914),  also  Jour.  Biol.  Chem.  17,  pp.  191,  211 


5.  Schunck:     Proc.  Roy.  Soc.  72,  p.  165  (1903). 

6.  Thudichum:     Proc.  Roy.  Soc.  17,  p.  253  (1869). 

7.  Willstatter  and  Escher:     Zeit.  f.  Physiol.  Chem.  76,  pp.  214-225 
(1912). 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  44! 


B,     CAROTIN  AND  XANTHOPHYLLS  DURING  DIC-ESTION 

The  establishment  by  us  l  of  both  a  chemical  and  physiological 
relation  between  the  carotin  and  xanthophylls  of  plants  and  the  yellow 
lipochromes  of  the  milk  fat,  body  fat,  blood  serum  and  corpus  luteum 
of  the  cow  has  shown  that  it  is  the  carotin  that  is  by  far  the  more 
important  in  pigmentation  of  the  animal  body.  It  is  a  well-known 
fact  that  xanthophylls  are  as  abundantly  and  sometimes  more  abun- 
dantly distributed  in  vegetable  matter  than  carotin.  The  question 
naturally  arises  then,  why  carotin  is  the  pigment  which  is  principally 
taken  up  by  the  cow's  body,  and  why  the  xanthophylls  appear  there 
only  in  very  small  quantity.  This  seemed  to  us  to  be  an  important 
physiological  question. 

It  will  readily  be  recognized  that  a  question  of  this  nature  is  not 
easily  answered.  It  may  therefore  be  stated  in  advance  that  the  results 
of  our  studies  were  not  as  satisfactory  as  was  anticipated.  The 
data  are  presented,  however,  for  what  value  they  may  possess,  since 
opportunity  was  not  presented  for  a  further  study  of  the  question. 
The  data  are  of  some  value,  at  least,  in  that  a  number  of  facts  are 
presented  which  are  sufficiently  related  to  advance  a  fairly  acceptable 
theory  in  regard  to  the  question. 

METHODS  OF  STUDY 

Several  methods  of  study  which  did  not  appear  to  offer  many 
difficulties,  seemed  available,  by  which  it  was  thought  light  could  be 
thrown  on  the  question.  One  method  was  to  study  the  action  of  the 
various  digestive  fluids,  both  natural  and  artificial,  on  fresh  crude 
residues  of  the  amorphous  carotin  and  xanthophylls  of  plants.  An- 
other method  was  to  study  the  nature  of  the  unsaponifiable  pigment 
extracts  at  various  places  along  the  digestive  tract  of  the  cow.  A 
third  method  was  closely  related  to  the  second  and  consisted  in  a  study 
of  the  unsaponifiable  yellow  pigments  excreted  under  conditions  where 
unassimilated  or  undestroyed  carotin  and  xanthophylls  of  the  food 
would  be  likely  to  appear  unchanged  in  the  feces.  Lack  of  time  made 
a  thorough  study  of  all  the  methods  impossible  so  that  only  the 
significant  features  of  the  results  of  each  study  will  be  given. 

1.     Missouri  Agricultural  Experiment  Station  Research  Bulletins  Nos.  10 
and  11,  this  Bulletin,  p.  415  (1914);  Jour.  Biol.  Chem.  pp.  191,  211,  223  (1914). 


442       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.  12 

THE  ACTION   OF   DIGESTIVE  JUICES 

The  following  solutions  were  added  to  equal  portions  of  carotin 
and  xanthophylls  *  in  test  tubes,  and  the  tubes  plugged  with  cotton 
and  set  aside  at  40° C.  Observations  for  signs  of  decomposition  were 
made  every  day  for  five  days. 

Tube  i.     Five  cc.  of  0.25  per  cent  HC1  solution  of  pepsin. 

Tube  2.  Five  cc.  of  0.25  per  cent  HC1  solution  of  filtered  gastric 
juice  from  the  fourth  stomach  of  a  Jersey  cow. 

Tube  3.     Five  cc.  of  0.25  per  cent  Na2CO3  solution  of  trypsin. 

Tube  4.  Five  cc.  of  0.25  per  cent  Na2CO3  solution  of  extract 
from  pancreas  of  a  Jersey  cow. 

Tube  5.  Five  cc.  of  0.25  per  cent  Na2CO3  solution  of  trypsin 
plus  5  cc.  of  fresh  bile  from  a  Jersey  cow. 

Tube  6.  Five  cc.  of  0.25  per  cent  NaCO  solution  of  pan- 
creatic extract2  plus  5  cc.  of  fresh  bile. 

1.  The  carotin  and  xanthophylls  were  isolated  as  follows:   200  grams  of 
air-dried,  powdered,  green  alfalfa  leaves  were  shaken  with  three  litres  of  10 
per  cent  alcoholic  petroleum  ether  for  two  days,  and  then  with  1  litre  of 
CS2,  until  the  solvent  had  taken  up  as  much  pigment  as  possible.    The  carotin 
and   xanthophylls    were    isolated    from    each    extract   and    combined.      Each 
solution  was  now  concentrated  to  50  cc.  and  divided  into  ten  parts.     These 
were  put  into  test  tubes  and  the  solvent  driven  off  at  a  low  temperature. 
The  residues  were  used  for  the  studies  reported  above. 

The  carotin  and  xanthophylls  were  isolated  from  the  alcoholic  petroleum 
ether  extract  as  follows:  The  xanthophylls  were  removed  from  the  extract 
by  shaking  with  an  equal  volume  of  80  per  cent  alcohol.  The  carotin  in 
the  petroleum  ether  was  now  freed  from  chlorophyll  by  shaking  with  an 
excess  of  CaCO3,  the  solution  was  now  evaporated  into  alcohol  and  trans- 
ferred to  ether  by  diluting  with  much  water  after  the  addition  of  ether. 
The  solution  was  freed  from  traces  of  chlorophyll  that  had  escaped  absorp- 
tion by  the  CaCO3  by  shaking  with  30  per  cent  alcoholic  potash.  The  ether 
was  then  freed  from  alkali  with  distilled  water.  This  ether  solution  of 
carotin  was  combined  with  the  similar  solution  obtained  from  the  CS2,  ex- 
tract as  described  below.  The  80  per  cent  alcohol,  containing  the  xantho- 
phylls, was  partially  freed  from  chlorophyll  by  shaking  with  moist  animal 
charcoal  for  one  hour.  The  pigments  were  then  transferred  to  ether,  the 
remainder  of  the  chlorophyll  being  removed  by  30  per  cent  alcoholic  potash 
as  in  the  case  of  the  carotin.  The  ether  solution  was  then  washed  free 
from  alkali  and  added  to  the  xanthophylls  obtained  from  the  CS2  extract 
as  described  below. 

The  carotin  and  xanthophylls  were  isolated  from  the  CS2  extract  as  follows: 
The  extract  was  concentrated  into  95  per  cent  alcohol  and  after  filtering  was 
saponified  with  KOH.  The  pigments  were  extracted  from  the  soap  with 
ether.  The  ether  was  washed  free  from  alkali  and  evaporated  into  alcohol. 
The  carotin  and  xanthophylls  were  separated  by  differentiation  between 
petroleum  ether  (b.  p.  30-50  °C.)  and  the  alcohol. 

2.  The  pancreatic  extract  was  prepared  by  extracting  a  freshly  ground 
cow's  pancreas  with  150  cc.  of  30  per  cent  alcohol  for  24  hours,  straining 
off  the  extract,  filtering    and    neutralizing    with    KOH    and    0.5    per    cent 
Na£CO3.     To  prepare  the  above  solution  an  equal  volume  of  0.5  per  cent 
Na2CO3  was  added. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  443 

Tube  7.     Five  cc.  of  neutral  solution  of  pancreatin. 

Tube  8.     Five  cc.  of  neutral  pancreatic  extract. 

Tube  9.  Five  cc.  of  neutral  pancreatic  solution  plus  5  cc.  of  fresh 
bile. 

Tube  10.     Five  cc.  of  neutral  pancreatic  extract  plus  5  cc.  of  bile. 

The  pepsin,  trypsin  and  pancreatin  were  Merck's  U.  S.  P.  prepara- 
tions. 

A  set  of  ten'  tubes  was  also  prepared  containing  equal  portions  of 
the  xanthophylls  of  yellow  corn.1 

The  following  results  were  obtained.  Carotin:  Bleaching  oc- 
curred only  in  the  tubes  containing  neutral  and  alkaline  pancreatic 
extracts.  In  the  same  tubes  plus  bile  there  was  no  decoloration. 
The  bile  had  no  solvent  action  on  the  carotin,  which  was  in  marked 
contrast  to  the  xanthophylls,  as  noted  below.  Xanthophylls :  The  pig- 
ments in  tubes  I,  3  and  4  were  largely  decolorized  at  the  end  of  the 
second  day,  while  those  in  tubes  2,  7  and  8  retained  their  color  after 
the  fifth  day.  No  observations  could  be  made  on  the  tubes  containing 
bile  until  the  fifth  day  on  account  of  the  fact  that  the  bile  had  com- 
pletely dissolved  the  pigments  as  soon  as  it  was  added.  The  pigments 
were  examined  by  desiccating  the  contents  of  the  tubes  with  plaster 
of  Paris  and  extracting  with  ether.  Marked  bleaching  had  occurred 
in  all  the  bile  tubes.  Corn  Xanthophylls :  There  was  marked  destruc- 
tive action  of  these  pigments  in  all  the  tubes  except  those  containing 
bile.  The  corn  xanthophylls,  like  the  xanthophylls  from  the  alfalfa, 
were  readily  soluble  in  bile. 

The  most  significant  feature  of  the  above  results  is  the  marked 
difference  in  the  solubility  of  carotin  and  xanthophylls  in  bile,  the 
surprising  result  being  the  very  slight  solubility  of  the  carotin.  This 
was  confirmed  quantitatively  using  carotin  from  another  source  and 
the  bile  from  several  different  cows.  The  results  are  given  in  Table  i. 
The  carotin  used  was  a  freshly  prepared  ether  solution  of  carotin  from 
the  carrot.  Equal  volumes  of  this  solution  were  evaporated  at  a  low 
temperature  and  the  residues  treated  with  10  cc.  of  bile  from  each  of 
four  cows.  After  standing  for  several  days  with  frequent  shaking 
the  bile  was  filtered  and  5  cc.  of  the  filtrate  desiccated  with  plaster 
of  Paris.  This  was  extracted  with  ether  until  colorless.  The  extract 
in  each  case  was  concentrated  to  a  low  volume,  made  up  to  12.5  cc. 
with  absolute  alcohol,  and  the  color  of  the  solution  measured  in  the 
Lovibond  Tintometer. 


1.     This   was  the   unsaponifiable   pigment   of   the   corn   which   was   more 
soluble  in  80  per  cent  alcohol  than  in  petroleum  ether. 


444       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.  12 
TABLE  No.  1. — THE  SOLUBILITY  OF  CAROTIN  IN  BILE. 


Experi- 

Source 

Carotin  used 

Carotin  in  bile 

Blank* 

ment 

of 

No. 

bile 

Yellow 

Red 

Yellow 

Red 

Yellow 

Red 

1. 

Jersey 

57.0 

2.0 

3.0 

0.6 

1.0 

0.2 

2. 

Angus 

57.0 

2.0 

9.0 

0.8 

1.0 

0.2 

3. 

Holstein 

57.0 

2.0 

10.0 

0.9 

1.0 

0.2 

4. 

Holstein 

57.0 

2.0 

10.5 

1.0 

1.0 

0.2 

*The  blank  is  the  amount  of  color  extracted   from   5   c.c,   of  bile  alone, 
after  desiccation  with  plaster  of  Paris. 

An  interesting  feature  in  the  above  table  is  the  apparent  greater 
solubility  of  carotin  in  the  bile  of  HJolstein  cows,  than  in  the  bile  of 
Jersey  cows.  If  this  is  confirmed  by  future  study,  considerable  sig- 
nificance could  be  attached  to  it  in  explaining,  at  least  partly,  the 
differences  between  the  two  breeds  in  the  amount  of  carotin  that  is 
secreted  in  the  milk  fat. 


CHARACTER  OF  THE  PIGMENTS  ALONG  THE  DIGESTIVE  TRACT 

The  plan  in  this  part  of  the  study  was  to  examine  the  pigments 
which  could  be  extracted  from  the  material  at  various  places  along  the 
digestive  tract  of  several  cows.  Material  was  obtained  from  one  Hol- 
stein cow  and  two  Jersey  cows  at  slaughtering,  from  each  of  the  three 
stomachs  just  before  the  food  entered  the  next  part  of  the  digestive 
tract,  from  three  places  in  the  small  intestines,  from  the  caecum,  and 
from  the  large  intestine.  One  or  two  hundred  grams  of  material  were 
either  dried  on  the  steam  bath  or  desiccated  with  plaster  of  Paris,  and 
the  resulting  mass  in  either  case  extracted  with  CS2.  The  solubility, 
spectroscopic,  and  adsorption  properties  of  the  extracted  pigments  were 
carefully  noted.  The  pigments  were  thus  differentiated  into  carotin 
and  xanthophyll  constituents  as  well  as  classified  as  belonging  to 
either  of  the  two  groups. 

The  results  of  the  study  were  not  satisfactory,  in  that  there  was 
no  uniformity  among  the  several  cows  in  regard  to  the  character 
of  the  pigments  found  at  any  particular  place,  although  all  the  animals 
were  receiving  a  ration  which  should  have  furnished  an  excess  of  both 
carotin  and  xanthophylls.  The  reason  for  this  is  not  obvious.  It 
might  be  thought  that  the  partial  drying  in  some  cases  destroyed  the 
pigments.  Possibly  this  occurred  to  some  extent,  but  it  would  not 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  445 

account  for  the  lack  of  uniformity  where  this  method  of  desiccation 
was  not  employed. 

No  further  discussion  will  be  given  this  study.  Mention  has  been 
made  of  it  merely  because  the  method  seems  to  be  a  valuable  one, 
and  will  warrant  further  application. 

THE    EXCRETED    PIGMENTS 

For  this  study  the  feces  of  a  cow  were  examined,  in  a  feeding- 
experiment  where  the  carotin  and  xanthophylls  were  furnished  by 
the  feeding  of  carrots  only.  The  balance  of  the  ration  was  composed 
of  grain  and  timothy  hay  almost  free  from  carotin  and  xanthophylls. 

The  method  of  demonstrating  the  character  of  the  pigments 
in  the  feces  was  to  desiccate  a  quantity  of  fresh  feces  with  plaster 
of  Paris  and  extract  the  mass  with  pure  carbon  bisulphide.  The  ex- 
tract was  concentrated  and  studied  spectroscopically,  and  also  by  means 
of  a  Tswett  chromotogramm.  The  relative  solubility  properties  of  the 
pigments  thus  found  were  studied,  and  also  the  spectroscopic  properties 
of  the  pigments  thus  separated. 

In  this  way  it  was  found  that  when  the  cow  was  receiving  50 
pounds  of  carrots  per  day,  both  carotin  and  xanthophylls  were  abun- 
dantly present  in  the  feces.  This  continued  for  six  days  after  the 
carrots  were  withdrawn  from  the  ration,  although  it  was  possible 
to  detect  but  little  xanthophyll  during  this  time. 

DISCUSSION  OF  RESULTS 

Combining  the  results  of  the  above  experiments,  the  appearance 
of  carotin  in  the  cow's  system  when  fed  in  excess  may  be  explained 
on  the  ground  of  its  greater  stability  toward  the  digestive  processes, 
as  shown  by  the  digestion  experiments,  and  the  abundant  appearance 
of  the  pigment  in  the  feces.  The  failure  of  the  xanthophylls  to 
appear  to  any  extent  in  the  cow's  system  may  be  due  similarly  to  the 
fact  that  they  are  apparently  more  easily  destroyed  1  during  digestion. 
Some  of  them  that  escape  destruction  are  undoubtedly  taken  up  by 
the  bile  and  thus  enter  the  system  through  the  portal  circulation. 
Some  oxidation  probably  takes  place  in  the  liver.  If  fat  is  present 
to  any  extent  some  of  the  xanthophylls  will  evidently  be  taken  up  and 

1.  Willstatter  and  Mieg.  (Ann.  d.  Chem.  355,  p.  1,  1907),  state  that 
xanthophylls  are  very  sensitive  toward  acids.  This  would  lead  one  to 
expect  that  they  would  be  largely  destroyed  by  the  gastric  juice.  Our 
results  were  contradictory  in  this  respect.  We  found  an  artificial  gastric 
juice  to  destroy  the  xanthophylls  but  the  natural  gastric  juice  from  the 
fourth  stomach  of  a  cow  apparently  had  no  effect  on  them. 


446       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.  12 

enter  the  blood  dissolved  in  fat.  In  this  connection  it  is  of  interest 
to  recall  that  we  have  shown  1  that  there  is  evidence  to  indicate  that 
what  xanthophylls  can  be  found  in  the  blood  are  present  dissolved 
in  fat. 

An  additional  possible  explanation  of  this  whole  question  should 
not  be  overlooked,  however,  namely,  that  the  difference  in  the  pro- 
portion of  carotin  and  xanthophylls  taken  up  by  the  cow's  body 
may  be  due  entirely  to  the  difference  in  chemical  composition 
between  carotin  and  xanthophylls.  Carotin  is  an  unsaturated  hydro- 
carbon and  is  furthermore  capable  of  combining  with  a  protein  of  the 
blood,  as  we  have  shown.2  The  xanthophylls,  on  the  other  hand,  are 
carbon,  hydrogen  and  oxygen  compounds,  in  fact  are  chemically  caro- 
tin-dioxides. Although  still  unsaturated  bodies,  their  slight  difference 
in  composition  from  carotin,  may  prevent  their  combination  with  the 
serum  albumin,  thus  making  it  impossible  for  them  to  appear  to  any 
extent  in  the  blood  and  fatty  formations  of  the  cow's  body.  If  fat 
played  a  greater  part  in  the  food  of  the  cow,  the  xanthophylls  would 
undoubtedly  appear  to  a  greater  extent  in  the  body  of  this  animal. 

SUMMARY 

1.  Carotin  is  assimilated  from  the  food  of  the  cow  in  preference 
to   xanthophylls   partly   because   of   its   greater   stability   toward   the 
juices  of  the  digestive  tract.     Xanthophylls  are  much  more  soluble 
in  bile  than  carotin,3  which  probably  accounts  for  their  appearance 
in  the  fat  of  the  blood. 

2.  It  is  probable  that  carotin  forms  by  far  the  greater  part  of 
the  lipochromes  of  the  cow's  body  chiefly  on  account  of  its  ability 
to   form  a  compound  with  one  of  the  proteins  of  the  blood.     The 
xanthophylls,  being  of  different  composition,  probably  are  not  capable 
of  forming  such  a  compound. 

BIBLIOGRAPHY 

1.  Fischer  and  Rose:     Zeit.  f.  Physiol.  Chem.  88,  p.  331  (1913). 

2.  Palmer  and  Eckles:     Missouri  Agricultural  Experiment  Station 
Research  Bulletins  Nos.   10  and   n    (1914);  Jour.  Biol.   Chem. 
17,  pp.  191,  211,  223  (1914). 

3.  Willstatter  and  Mieg:  Ann.  d.  Chem.  355,  p.  I   (1907). 

1.  This  Bulletin,  page  422;   Jour.  Biol.  Chem.  17,  p.  211,  1914. 

2.  lUd. 

3.  A  confirmation  of  the  very  slight  solubility  of  carotin  in  bile  is  seen 
in  the  recent  finding  of  Fischer  and  Rose  (Zeit  f.  Physiol.  Chem.  88,  p.  331, 
1!913),    that    the   gall    stones    of    cows    contain    crystallizable    carotin.      No 
xanthophylls  were  found. 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  447 


C.     THE  PIGMENTS  OF  HUMAN  MILK  FAT 

The  discovery  of  the  chemical  and  physiological  relations  of 
the  pigments  of  the  fat  of  cow's  milk  to  the  carotin  and  xanthophylls 
of  plants  naturally  opens  the  question  whether  the  pigments  which 
characterize  the  fat  of  other  animals  are  of  a  similar  character. 
Opportunity  was  not  afforded  to  study  this  question  with  any  domestic 
animals  other  than  the  cow.  An  opportunity,  however,,  was  offered 
to  investigate  the  character  of  the  pigments  which  sometimes  give  a 
high  color  to  the  fat  of  human  milk. 

The  methods  used  for  studying  the  character  of  the  pigments 
were  the  microscopic  ones  used  in  the  preceding  studies.  The  adsorp- 
tion properties  were  not  studied,  however,  the  demonstration  being 
confined  to  the  observation  of  the  absorption  bands  and  the  relative 
solubility  properties. 

The  fat  from  two  samples  of  human  milk  from  different  sources 
was  used.  Very  little  was  known  in  regard  to  one  of  the  samples, 
it  having  been  sent  to  the  laboratory  for  analysis  by  a  well-known 
physician  of  the  community.  The  other  sample  was  taken  by  one  of 
us  from  a  woman  who  had  just  given  birth  to  a  child,  and  represented 
a  portion  of  the  milk  of  each  day  of  the  first  few  days  of  lactation. 
Some  further  observations  in  regard  to  this  sample  will  be  reported 
below. 

Experiment  No.  i 

This  was  the  sample  in  regard  to  which  very  little  was  known, 
with  the  exception  that  it  was  a  bona  fide  sample  of  human  milk. 
The  milk  had  a  faint  yellow  tint.  The  volume  of  milk  used  was 
approximately  125  c.cm.  The  milk  contained  about  3.5  per  cent  fat 
and  therefore  yielded  a  little  over  4  grams  of  fat.  The  fat  was 
obtained  from  the  milk  by  precipitating  it  along  with  the  proteins. 
To  do  this  the  milk  was  acidified  with  acetic  acid,  a  pinch  of  salt 
added,  and  the  milk  brought  to  a  boil.  The  precipitated  proteins, 
when  filtered  off,  had  a  bright  yellow  color,  due  to  occluded  fat. 
The  fat  was  dissolved  out  with  hot  95  per  cent  alcohol. 

After  concentrating  the  alcoholic  extract,  the  fat  was  saponified 
by  adding  a  small  piece  of  KOH  and  boiling  for  about  one  hour. 
The  pigment  was  readily  extracted  from  the  soap  by  ether,  after 
dilution  with  water.  '  The  golden-yellow  ether  solution  was  washed 
with  water  and  evaporated  to  dryness.  The  residue  dissolved  at  once 
in  carbon  bisulphide  with  a  red-orange  color  and  in  this  solution 


448       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.  12 


showed  two  beautiful  absorption  bands,  and  possibly  a  third.  The 
CS2  was  carefully  evaporated.  A  part  of  the  residue  which  remained 
was  difficultly  soluble  in  absolute  alcohol,  but  readily  dissolved  when 
a  little  petroleum  ether  was  added.  When  differentiated  between 
petroleum  ether  and  80  per  cent  alcohol  the  combined  pigment  was 
readily  divided  into  two  apparently  equal  proportions  with  perhaps 
slightly  more  color  in  the  petroleum  ether  layer. 

The  pigment  of  the  petroleum  ether  layer  gave  a  red-orange 
carbon  bisulphide  solution  showing  two  strong  absorption  bands  and 
a  third  faint  one,  the  measurements  of  which  are  given  in  Table  No.  i 
below.  j 

TABLE  No.  1. — ABSORPTION  BANDS  OF  CAROTIN  AND  XANTHOPHYLLS  FROM 
HUMAN    MILK   FAT. 


Experi- 
ment. 

Measurements  of  absorption  bands. 

Carotin 

Xanthophylls. 

No.  1 

I. 
II. 
III. 

225—244 
262—280 
300—319 

I.     234—253 
II.     275—293 
III.     320— 

No.  2 

I. 
II. 
III. 

225—242 
265—282 

I.     232—252 
II.     273—293 
III.     312—330 

The  pigment  of  the  alcoholic  layer  gave  a  yellow-orange,  carbon 
bisulphide  solution  showing  two  good  absorption  bands  and  end  absorp- 
tion, the  measurements  of  which  are  given  in  Table  i. 

Experiment  No.  2 

As  stated  above,  this  sample  of  human  milk  was  taken  by  one 
of  us  and  represented  the  milk  of  the  first  few  days  of  lactation 
including  the  colostrum  milk.  The  milk  itself  was  characterized 
by  a  high  yellow  color  and  the  fat  which  rose  to  the  top  of  the 
sample  had  a  very  deep  yellow  color.  About  350  c.cm.  of  milk  were 
obtained.  The  fat  percentage  being  between  5  and  6,  nearly  20 
grams  of  fat  were  yielded  for  the  study  of  the  pigments. 

The  fat  was  obtained  from  this  sample  of  milk  in  a  manner 
very  similar  to  that  used  in  the  preceding  experiment.  The  proteins 
and  fat  were  precipitated  together  by  adding  a  little  salt  and  also  con- 


CAROTIN,  THE  PRINCIPAL  YELLOW  PIGMENT  OF  MILK  FAT  449 

siderable  ammonium  sulphate,  acidifying  with  acetic  acid  and  bringing 
to  a  boil.  The  precipitate  was  filtered  off  on  a  Bikhner  funnel.  The 
layer  of  protein  and  fat  had  a  golden-yellow  color.  The  fat  was 
extracted  with  hot  alcohol  and  ether.  The  golden-colored  extract 
was  evaporated  to  dryness  and  the  fat  dissolved  away  with  ether. 
Alcohol  was  added  and  also  5  grams  of  KOH  and  saponification  of 
the  fat  allowed  to, proceed  on  the  steam  bath  for  one-half  hour.  The 
pigment  was  extracted  from  the  diluted  soap  with  ether.  After 
thorough  washing  with  distilled  water,  the  ether  was  evaporated  care- 
fully to  dryness.  The  residue  had  a  deep  red  color.  It  dissolved 
at  once  in  petroleum  ether  (b.p.  3O°-5o°C.). 

The  pigment  in  this  solution  was  now  differentiated  between  the 
petroleum  ether  and  80  per  cent  alcohol.  In  this  way  it  was  divided 
into  two  portions  which  were  about  equal  as  far  as  could  be  detected 
by  the  color  of  the  two  solutions,  with  perhaps  slightly  more  color 
in  the  80  per  cent  alcohol. 

The  pigment  in  the  petroleum  ether  layer  gave  a  blood-red  colored 
carbon-bisulphide  solution  which  showed  two  absorption  bands  and 
considerable  end  absorption.  The  measurements  of  these  bands  are 
given  in  Table  i. 

The  pigment  in  the  80  per  cent  alcohol  layer  gave  an  orange- 
colored  carbon-bisulphide  solution  which  showed  three  distinct  absorp- 
tion bands.  The  measurements  of  these  bands  are  given  in  Table  No.  I. 

DISCUSSION  OF  RESULTS 

The  results  of  the  above  experiments  show  very  clearly  that  the 
fat  of  human  milk  may  be  tinted  with  the  same  pigments  found  in 
the  fat  of  cow's  milk.  The  relative  proportion  of  carotin  and 
xanthophylls  in  human  milk  fat  is  much  more  nearly  equal  than  in 
the  fat  of  cow's  milk.  This  is  not  surprising  when  it  is  considered 
that  there  is  strong  evidence  that  the  xanthophylls  are  'conveyed 
through  the  body  dissolved  in  fat,  and  when  it  is  also  considered 
that  fat  plays  a  much  greater  part  in  human  food  than  in  the  food 
of  the  cow.  i 

An  especially  interesting  fact  brought  out  by  these  brief  studies 
is  that  colostrum  milk  fat  of  the  human  is  characterized  by  a  very 
high  color  just  as  is  the  case  with  the  fat  of  the  colostrum  milk 
from  cows.  In  the  experiment  here  reported,  one  of  us  had  occasion 
to  observe  that  after  about  ten  days  the  milk  fat  from  the  same  woman 
was  very  much  lighter  in  color  than  during  the  first  few  days  of 
lactation.  The  milk  was  also  observed  at  intervals  for  a  period  of 


45O       MISSOURI  AGRICULTURAL  EXP.  STA.  RESEARCH  BULLETIN  NO.  12 

several  months.  Considerable  variation  in  the  color  of  the  fat  was 
noticed.  Although  it  was  not  possible  to  accurately  trace  the  cause 
of  this  variation,  as  we  did  in  the  case  of  cows  in  an  earlier  paper 
of  this  series,  it  was  undoubtedly  due  to  changes  in  diet. 

In  conclusion  it  may  be  stated  that  all  students  of  human  anatomy 
are  familiar  with  the  fact  that  the  fat  on  the  human  body  is  often 
characterized  by  a  marked  yellow  color.  In  view  of  the  fact  that  the 
pigments  of  the  milk  fat  and  body  fat  of  the  cow  are  identical,  it 
must  therefore  be  concluded  that  the  pigments  of  the  milk  fat  and 
body  fat  of  humans  are  identical. 

SUMMARY* 

1.  The  fat  of  human  milk  may  be  tinted  by  carotin  and  xantho- 
phylls,  the  pigments  which  characterize  the  fat  of  cows'  milk.     The 
relative  proportion  of  carotin  to  xanthophyll  in  human  milk   fat  is 
much  more  nearly  equal  than  in  the  fat  of  cows'  milk. 

2.  The  colostrum  fat  of  human  milk  is  characterized  by  a  very 
high  color  as  is  the  case  with  the  fat  of  the  colostrum  milk  of  cows. 

3.  The  pigment  of  human  body  fat  is  no  doubt  identical  with  the 
pigment  of  human  milk  fat. 

*See  page  438  for  summary  of  "The  Yellow  Pigment  of  Blood  Serum." 
See  page  446  for  summary  of  "Carotin  and  Xanthophylls  During  Digestion." 


BIOGRAPHY 

Leroy  Sheldon  Palmer  was  born  in  Rushville,  Illinois,  on  March 
23,  1887.  He  received  his  common  school  education  in  the  public 
schools  of  the  city  of  St.  Louis,  Missouri,  graduating  from  the  Central 
High  School  of  that  city  in  June,  1905.  He  entered  the  School  of 
Engineering  of  the  University  of  Missouri  in  September,  1905,  and 
received  the  degree  of  B.S.  in  Chemical  Engineering  in  June,  1909. 
During  the  summer  of  1909  he  was  Chemical  Assistant  for  the  United 
States  Bureau  of  Fisheries,  the  work  being  conducted  under  the  direc- 
tion of  Dr.  Chas.  W.  Greene  at  Columbia,  Missouri.  He  was  ap- 
pointed Fellow  in  Chemistry  at  the  University  of  Missouri  for  the 
year  1909-1910,  but  resigned  in  October,  1909,  to  become  Assistant 
Chemist  in  the  Co-operative  Government  Dairy  Research  Laboratory 
of  the  University  of  Missouri,  being  appointed  by  the  Dairy  Division 
of  the  Bureau  of  Animal  Industry  of  the  United  States  Department 
of  Agriculture.  He  pursued  graduate  work  in  the  University  of  Mis- 
souri during  the  years  1909-1910  and  1910-11,  and  received  the  degree 
of  M.A.  in  June,  1911.  He  was  appointed  by  the  Dairy  Division  of 
the  United  States  Department  of  Agriculture  in  October,  1911,  as  the 
Government  Representative  in  the  Co-operative  Dairy  Research  Lab- 
oratory of  the  University  of  Missouri.  He  pursued  work  in  the 
Graduate  School  of  the  University  of  Missouri  during  1911-1912  and 
1912-13.  He  was  appointed  Assistant  Professor  of  Dairy  Chemistry 
and  Assistant  Chemist  to  the  Experiment  Station  in  the  Department  of 
Agricultural  Chemistry  by  the  University  of  Missouri  in  April,  1913. 


451 


LD  21-95m-7,'37 


/ 


7*3 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


